Nature Genetics: doi: /ng Supplementary Figure 1. Parameters and consequences of mononuclear cardiomyocyte frequency.

Transcription

1 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 See Supplementary Table 1 for a full list of strains and values. (be) 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 < 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.

2 Supplementary Figure 2 Relationships of Tnni3k to mononuclear cardiomyocyte frequency.

3 (a) Dot plot of 120 HMDP strains distributed across three alleles of Tnni3k. Allele 1 is a C at rs resulting in a splice mutation and reduced expression (n = 60 strains). Allele 2 is a T at rs with wild-type expression (n = 56 strains). Allele 3 is T at rs 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.

4 Supplementary Table Strains of the Hybrid Mouse Diversity Panel. # on Strain N Average Biological Replicates Average SEM Fig1A %Mono %Multi SEM 1 AXB24/PgnJ % 0.74% 0.83% 3.06% 3.06% 11.38% 1.46% 2 BXD24b/TyJ % 0.19% 2.68% 2.29% 7.00% 1.04% 3 BXD31/TyJ % 0.54% 3.67% 2.55% 1.81% 8.76% 2.63% 4 SJL/J % 0.50% 3.66% 1.95% 2.70% 21.48% 2.35% 5 BXD29/TyJ % 1.03% 3.82% 1.77% 10.41% 0.21% 6 BXH6/TyJ % 0.34% 2.86% 2.24% 3.44% 18.01% 1.51% 7 C58/J % 0.60% 3.41% 3.51% 1.66% 9.20% 1.88% 8 BXH8/TyJ % 0.68% 3.33% 3.86% 1.60% 16.53% 1.62% 9 BXA24/PgnJ % 0.38% 3.54% 2.79% 5.34% 1.35% 10 BXD85/RwwJ % 0.04% 3.23% 3.13% 6.10% 1.22% 11 BXA26/PgnJ % 0.85% 2.52% 4.21% 2.97% 0.87% 12 BXD34/TyJ % 0.61% 2.47% 3.26% 4.55% 4.40% 0.36% 13 C57BLKS/J % 0.89% 3.72% 1.76% 4.79% 8.15% 0.96% 14 BTBR T+ tf/j % 0.62% 3.13% 2.55% 4.62% 9.29% 1.76% 15 BXD5/TyJ % 1.01% 2.08% 3.13% 5.49% 9.09% 2.61% 16 MRL/MpJ % 1.30% 2.43% 6.21% 2.17% 5.08% 0.15% 17 BXD75/RwwJ % 0.75% 4.71% 1.49% 4.12% 4.56% 8.46% 1.37% 18 BXD16/TyJ % 0.25% 3.33% 4.21% 3.72% 3.45% 0.71% 19 BXD67/RwwJ % 1.03% 5.19% 2.67% 8.33% 0.09% 20 AXB10/PgnJ % 0.21% 4.19% 3.77% 11.20% 0.50% 21 AXB19/PgnJ % 0.85% 3.39% 4.71% 2.25% 6.18% 9.08% 0.42% 22 BXD96/RwwJ % 0.84% 5.70% 2.67% 2.75% 5.51% 7.57% 0.95% 23 BXH10/TyJ % 0.79% 4.92% 2.70% 5.21% 16.75% 0.33% 24 AXB15/PgnJ % 0.23% 4.66% 3.87% 4.39% 4.01% 0.08% 25 BXA7/PgnJ % 1.44% 2.05% 6.08% 2.56% 10.04% 1.57% 26 BXD18/TyJ % 0.31% 5.04% 4.05% 4.15% 8.72% 2.56% 27 BXH22/KccJ % 0.85% 2.87% 5.76% 4.72% 6.37% 0.32% 28 AXB12/PgnJ % 1.12% 3.33% 5.58% 3.23% 0.64% 29 BXD87/RwwJ % 0.43% 4.39% 5.24% 3.76% 16.02% 3.15% 30 BXD65/RwwJ % 0.27% 4.80% 3.95% 4.72% 8.55% 0.47% 31 BXA14/PgnJ % 0.90% 3.61% 5.41% 9.72% 0.59% 32 SM/J % 0.76% 5.51% 3.65% 6.47% 2.67% 33 BXH9/TyJ % 1.04% 5.47% 2.54% 5.81% 8.10% 2.59% 34 BXD55/RwwJ % 0.78% 5.17% 4.12% 6.45% 2.80% 20.57% 3.49% 35 BUB/BnJ % 0.77% 3.57% 4.41% 6.18% 5.91% 0.84% 36 CXB6/ByJ % 1.42% 3.33% 6.17% 19.98% 4.71% 37 BXD51/RwwJ % 0.23% 4.52% 5.31% 4.76% 6.31% 1.32% 38 AXB19a/PgnJ % 0.33% 5.43% 4.29% 5.00% 10.16% 1.35% 39 FVB/NJ % 0.39% 4.64% 6.08% 4.44% 4.52% 9.52% 1.67% 40 BXD32/TyJ % 0.62% 6.28% 4.48% 4.37% 8.06% 1.22% 41 BXD73/RwwJ % 0.64% 4.70% 6.94% 4.81% 3.95% 22.79% 1.85% 42 BXD2/TyJ % 0.73% 4.38% 5.84% 3.45% 1.41% 43 C57Bl/6J % 0.50% 4.65% 4.49% 6.67% 5.03% 20.28% 5.66% 44 AXB23/PgnJ % 0.29% 5.06% 5.65% 12.13% 1.58% 45 C57L/J % 0.77% 4.15% 5.16% 6.78% 7.73% 1.10% 46 BXD60/RwwJ % 2.51% 2.33% 8.47% 6.90% 13.31% 5.33% 47 SEA/GnJ % 0.61% 4.55% 5.26% 6.61% 5.93% 1.63% 48 BXH4/TyJ % 0.52% 5.58% 6.10% 4.00% 6.28% 15.49% 1.27% 49 BXD14/TyJ % 0.41% 5.62% 6.16% 4.76% 8.24% 1.36% 50 BXD40/TyJ % 0.28% 5.91% 5.78% 5.00% 6.38% 1.70% X1/SvJ % 1.12% 6.42% 6.98% 3.37% 6.04% 0.58% 52 CXB8/HiAJ % 1.20% 6.43% 2.83% 5.05% 9.76% 3.88% 14.47% 2.29% 53 BXH19/TyJ % 1.51% 5.05% 8.53% 3.40% 9.98% 0.57% 54 CXB9/HiAJ % 0.08% 5.74% 5.59% 10.36% 0.52% 55 BXD13/TyJ % 0.44% 6.49% 5.56% 5.00% 0.77% 0.49% 56 BXD6/TyJ % 1.87% 2.08% 3.13% 5.49% 11.19% 1.55% 57 CXB12/HiAJ % 0.23% 5.45% 6.23% 5.63% 6.08% 1.92% 58 Balb/cJ % 0.67% 4.80% 5.68% 7.11% 4.89% 0.58% 59 BXD28/TyJ % 0.55% 6.46% 4.89% 6.61% 4.69% 1.04% 60 KK/HlJ % 0.35% 6.78% 6.02% 5.59% 10.08% 2.67%

5 Supplementary Table 1. Continued # on Strain N Average Biological Replicates Average SEM Fig1A %Mono %Multi SEM 61 BXH7/TyJ % 1.36% 7.00% 7.96% 3.48% 4.91% 0.44% 62 CXB13/HiAJ % 0.30% 5.86% 6.46% 20.42% 0.75% 63 LG/J % 0.47% 6.64% 5.70% 10.26% 0.50% 64 AKR/J % 1.25% 4.88% 4.96% 8.68% 17.72% 1.24% 65 BXD61/RwwJ % 0.89% 4.50% 6.64% 7.49% 4.89% 0.29% 66 BXD68/RwwJ % 1.20% 5.09% 7.49% 3.36% 1.05% 67 LP/J % 0.65% 7.06% 5.75% 3.12% 1.30% 68 BXH20/KccJ % 0.81% 7.26% 5.65% 12.90% 1.61% 69 BXD50/RwwJ % 0.67% 7.59% 6.58% 5.26% 5.22% 1.20% 70 BXD20/TyJ % 0.75% 7.92% 7.42% 6.33% 7.45% 3.77% 13.51% 2.59% 71 BXD48/RwwJ % 0.56% 5.78% 8.00% 7.11% 5.65% 17.52% 3.06% 72 BXA2/PgnJ % 0.65% 6.00% 7.29% 18.38% 0.38% 73 BXA13/PgnJ % 0.34% 7.04% 7.20% 6.10% 9.76% 1.30% 74 BXD42/TyJ % 0.87% 5.49% 6.59% 8.47% 8.33% 1.54% 75 DBA/2J % 0.30% 7.54% 7.20% 6.51% 9.31% 1.16% 76 BXD9/TyJ % 0.90% 8.23% 6.43% 5.74% 1.88% 77 BXD100/RwwJ % 0.78% 6.64% 7.00% 9.13% 9.65% 0.89% 78 AXB1/PgnJ % 0.82% 8.81% 8.17% 6.08% 12.79% 2.22% 79 BXD27/TyJ % 0.26% 7.91% 8.28% 7.37% 4.23% 1.13% 80 NZW/LacJ % 1.18% 6.50% 9.39% 5.91% 0.16% 81 MA/MyJ % 1.10% 8.11% 8.68% 7.39% 5.65% 2.93% 82 BXA11/PgnJ % 0.18% 8.00% 8.44% 3.28% 0.26% 83 CBA/J % 0.24% 8.36% 8.66% 7.84% 7.41% 0.83% 84 BXD38/TyJ % 1.43% 10.93% 8.41% 5.98% 9.38% 1.76% 85 AXB2/PgnJ % 1.08% 8.28% 6.29% 10.65% 11.11% 5.90% 5.24% 1.10% 86 BXD1/TyJ % 1.99% 6.98% 12.50% 6.19% 2.91% 0.63% 87 BXD8/TyJ % 0.56% 9.20% 8.08% 4.03% 1.35% 88 BXA4/PgnJ % 1.19% 6.98% 9.52% 11.64% 6.56% 6.58% 2.29% 89 BXD69/RwwJ % 1.27% 11.34% 7.08% 8.30% 4.47% 0.08% 90 BXA12/PgnJ % 1.40% 6.14% 9.75% 7.55% 12.55% 3.87% 0.45% 91 BXD15/TyJ % 0.91% 7.57% 9.41% 10.71% 5.04% 0.72% 92 Nor/LTJ % 0.10% 9.55% 9.34% 9.67% 7.29% 0.60% 93 NOD/LtJ % 2.20% 6.99% 7.69% 13.92% 3.47% 1.45% 94 BXA25/PgnJ % 0.50% 9.15% 9.58% 8.98% 11.16% 12.74% 1.03% 95 CXB1/ByJ % 0.06% 9.78% 9.90% 4.20% 0.14% 96 RIIIS/J % 0.68% 10.84% 10.55% 9.31% 7.56% 11.34% 4.71% 1.52% 97 BXD39/TyJ % 1.14% 9.01% 12.23% 8.62% 2.17% 1.51% 98 AXB4/PgnJ % 0.26% 9.70% 10.23% 6.82% 1.13% 99 PL/J % 0.67% 9.28% 10.93% 6.40% 0.06% 100 AXB8/PgnJ % 1.33% 13.28% 9.91% 8.02% 10.15% 10.97% 13.24% 2.10% 101 BXH14/TyJ % 1.37% 13.00% 8.26% 10.81% 5.17% 0.68% 102 I/LnJ % 0.54% 9.73% 11.44% 11.22% 4.37% 1.80% 103 C3H/HeJ % 1.05% 13.64% 9.49% 8.98% 11.17% 7.22% 0.81% 104 BXD19/TyJ % 1.86% 7.59% 11.62% 13.94% 4.96% 0.96% 105 BXD21/TyJ % 1.39% 8.24% 16.31% 8.19% 12.98% 13.13% 8.53% 4.89% 0.95% 106 BXD11/TyJ % 1.95% 8.82% 10.14% 15.22% 2.88% 0.97% 107 CXB2/ByJ % 1.41% 11.65% 15.09% 8.28% 10.8% 3.64% 0.76% 108 BXD84/RwwJ % 1.54% 9.93% 13.02% 11.51% 0.92% 109 AXB6/PgnJ % 1.81% 11.98% 8.30% 14.54% 4.51% 0.92% 110 CXB11/HiAJ % 1.66% 9.09% 14.79% 11.30% 1.16% 0.20% 111 CE/J % 0.96% 13.23% 10.00% 12.37% 6.81% 0.70% 112 BXA16/PgnJ % 1.12% 13.64% 11.64% 10.51% 7.64% 1.71% 113 BXA8/PgnJ % 0.29% 12.55% 11.97% 4.32% 1.75% 114 AXB13/PgnJ % 3.59% 9.09% 16.28% 4.31% 1.98% 115 BXA1/PgnJ % 1.30% 11.63% 13.29% 17.92% 14.21% 9.34% 11.72% 8.17% 2.08% 116 Balb/cByJ % 1.48% 15.69% 16.88% 10.13% 13.40% 9.15% 1.93% 117 BXD12/TyJ % 2.84% 20.00% 11.16% 11.83% 3.23% 0.72% 118 AXB5/PgnJ % 0.18% 14.58% 14.13% 1.41% 0.58% 119 SWR/J % 1.14% 12.22% 17.54% 15.72% 16.33% 4.33% 1.42% 120 A/J % 1.98% 15.87% 12.92% 25.21% 13.93% 13.82% 9.38% 22.43% 22.22% 8.20% 1.43%

6 Supplementary Table 2. Statistics for Figure 1F. Ejection Fraction across 4 strains. ANOVA - Repeated Measures: P-value (interstrain): P = 0.11 P-value (intrastrain*day): P < INTERSTRAIN VARIATION: ANOVA - Baseline P = 0.94 ANOVA - 3 day P = 0.50 ANOVA - 28 day P = Bonferonni P-value Significant Bonferonni P-value Significant Bonferonni P-value Significant A vs SWR A vs SWR A vs SWR A vs C A vs C A vs C A vs SJL A vs SJL A vs SJL SWR vs C SWR vs C SWR vs C SWR vs SJL SWR vs SJL SWR vs SJL C57 vs SJL C57 vs SJL C57 vs SJL INTRASTRAIN VARIATION INTERSTRAIN VARIATION (low vs high strains) Paired T-tests (intrastrain: EF day 3 to day 28) T-Test (combined low strains vs combined high strains) Strain P-value Significant Time point P-value Significant A Baseline SWR trending 3 day C57Bl/ day SJL High strains combined Low strains combined 8.70E-04 Supplementary Table 3. Statistics for Figures S1B-E. Additional parameters of echocardiography across strains at 28 days post infarction. Figure S1B. LV Anterior Wall (28 day) Figure S1C. Stroke Volume (28 day) ANOVA - 28 day P = 3E-05 ANOVA - 28 day P = Bonferonni Correction P-value Significant Bonferonni Correction P-value Significant A vs SWR A vs SWR A vs C57Bl/ A vs C57Bl/ A vs SJL A vs SJL SWR vs C57Bl/ SWR vs C57Bl/ SWR vs SJL 1.1E-04 SWR vs SJL C57Bl/6 vs SJL C57Bl/6 vs SJL Figure S1D. LV Volume (systole, 28 day) Figure S1E. LV Volume (diastole, 28 day) ANOVA - 28 day P = 9E-04 ANOVA - 28 day P = 2E-04 Bonferonni Correction P-value Significant Bonferonni Correction P-value Significant A vs SWR A vs SWR A vs C57Bl/ A vs C57Bl/ A vs SJL A vs SJL SWR vs C57Bl/ SWR vs C57Bl/6 1.8E-04 SWR vs SJL SWR vs SJL C57Bl/6 vs SJL C57Bl/6 vs SJL 0.483

7 Full Western blot images corresponding to Figure 3C. Cropped off 3 A/J samples (irrelevant), #1969 (misloaded or degraded), and last two samples (unrelated experiment)

8 mou se mou heart cmlc se heart cmlc 2-GFP 2 cmlc -Tnni3k # 2-T 1 H2O nni3k #1 Full Western blot images corresponding to Figure S2B. 2 samples cropped after SWR (unrelated experiment) zebrafish - Tnni3k mtnni3k (primer set 2) mou se mou heart cmlc se heart cmlc 2-GFP 2 cmlc -Tnni3k # 2-T 1 H2O nni3k #1 mou se mou heart cmlc se heart cmlc 2-GFP 2 cmlc -Tnni3k # 2-T 1 H2O nni3k #1 mtnni3k (primer set 1) zebrafish - eef1g Full RT-PCR gel images corresponding to Figure S2J. Full Western blot images corresponding to Figure S2K. Anti-Tnni3k blot on left. Anti-Gapdh blot on right.