The impact of non-cardiomyocyte MYBPC3 expression on the development of hypertrophic cardiomyopathy

23

24 Hypertrophic Cardiomyopathy (HCM) is a disease defined by the development of left ventricle 25 hypertrophy. One of the most commonly mutated genes in HCM is cardiac myosin binding 26 protein C (MYBPC3). MYBPC3 protein localizes to the cardiomyocyte sarcomere, but studies 27 have reported detection of both MYBPC3 RNA and protein in non-cardiomyocyte cell 28 populations. Therefore, it was unclear if MYBPC3 expression in non-cardiomyocyte cell 29 populations altered the development of cardiomyopathy caused by MYBPC3 protein 30 deficiency. 31

32 We utilized genetically modified murine models with germline deletion of Mybpc3 exons 3 to 5 33 (Mybpc3-/-) or cardiomyocyte specific deletion of Mybpc3 exons 3 to 5 (Mybpc3fl/fl ; Myh6-Cre). 34 Gene expression was assessed using quantitative RT-PCR. Whole tissue protein levels were 35 assessed using immunoblots . Immunohistochemistry and proximity ligation assays were 36 performed to evaluate in situ protein expression. Echocardiography was utilized to measure 37 left ventricular structure and function. 38

39 Mybpc3 mRNA was detected in multiple organs including the heart, lung and blood from both 40 humans and mice. Utilizing transgenic murine models with germline or cardiomyocyte specific 41 deletion of Mybpc3 exons 3-5, we discovered that the Mybpc3 mRNA detected in extracardiac 42 locations originated primarily from cardiomyocytes. Likewise, MYBPC3 protein was identified 43 in myocardial tissue but not in other organs and cardiomyocytes were the only cell population 44 in myocardial tissue that had detectable MYBPC3 protein. Importantly, cardiomyocyte deletion 45 of Mybpc3 caused similar pathological myocardial remodeling and alterations in left ventricular 46 function compared to germline deletion of Mybpc3 in all cell populations. 47 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint

48 Our results show that cardiomyocytes are the primary cell source of Mybpc3 mRNA detected 49 in extracardiac organs and they are the principal cell type responsible for the cardiomyopathy 50 caused by MYBPC3 protein deficiency. These results suggest that selective targeting of 51 cardiomyocytes should be the most efficient approach to treat cardiomyopathies associated 52 with MYBPC3 deficiency. 53 54 Non-standard Abbreviations and Acronyms 55 HCM: hypertrophic cardiomyopathy 56 LV: left ventricle 57 LVH: left ventricular hypertrophy 58 MYBPC3: myosin binding protein C3 59 60

61 Hypertrophic cardiomyopathy (HCM) is characterized by the development of the left ventricle 62 hypertrophy and has a prevalence of at least 1 in 500 in humans .1 This disease is often 63 inherited, and the most common genetic causes of this condition are mutations in the 64 sarcomere proteins myosin heavy chain 7 ( MYH7) and cardiac myosin binding protein C 65 (MYBPC3).2 Mutations in MYH7 are typically thought to cause disease through a gain of 66 function mechanism .3,4 In contrast, mutations in MYBPC3 are primarily thought to cause 67 disease through a loss of function haploinsufficiency mechanism.5-7 68 MYBPC3 protein has been shown to be an integral component of the cardiomyocyte 69 sarcomere where it regulates the interaction of myosin and actin filaments to modulate 70 sarcomere contraction.7-9 However, MYBPC3 RNA has been detected in multiple extracardiac 71 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint tissues such as the blood , lung, adrenal gland , and skeletal muscle .10,11 Likewise, non-72 cardiomyocyte cells such as myocardial fibroblasts and the NIH-3T3 cell line were reported to 73 express MYBPC3 protein.12 Likewise, murine models deficient in MYBPC3 protein develop 74 myocardial hypertrophy that is associated with alterations in not only cardiomyocytes but also 75 non-cardiomyocyte cell populations.13,14 It was assumed that the changes detected in these 76 non-cardiomyocyte myocardial cell populations were secondary to the pathologic changes in 77 cardiomyocyte growth and function resulting from MYBPC3 protein deficiency. However, it 78 remained unclear whether non-cardiomyocytes expressed MYBPC3 protein and if this non -79 cardiomyocyte expression altered the development of cardiomyopathy caused by MYBPC3 80 protein deficiency. 81 Deciphering the impact of non -cardiomyocyte MYBPC3 protein expression is particularly 82 important since emerging methods to treat MYBPC3 related cardiomyopathies selectively 83 target the cardiomyocyte cell population.15,16 In order to address this question, we compared a 84 murine model with germline Mybpc3 deletion in all cells to a murine model with selective 85 cardiomyocyte Mybpc3 deletion. We used these in vivo models to identify the primary source 86 of extracardiac Mybpc3 RNA and to determine if non-cardiomyocyte M YBPC3 protein 87 expression impacts the development and progression of cardiomyopathy resulting from 88 MYBPC3 deficiency. 89

90 Cardiomyocytes are the primary source of extracardiac Mybpc3 mRNA 91 We utilized the Genotype Tissue Expression (GTEx) dataset to evaluate human MYBPC3 RNA 92 expression in cardiac and extracardiac tissues. We discovered that human MYBPC3 RNA 93 expression was highest in the heart but was also detected in other organs such as blood and 94 lung (Figure 1A). Similar to humans, mice also had Mybpc3 mRNA expression in extracardiac 95 tissues such as the lungs and blood (Figure 1B, S1A). To investigate the source of 96 extracardiac Mybpc3 mRNA, we utilized a transgenic murine model with germline deletion of 97 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint exons 3 to 5 of the Mybpc3 gene in all cell types (Mybpc3-/-) (Figure 1C) and a transgenic 98 murine model that eliminates exons 3 to 5 of the Mybpc3 gene specifically in cardiomyocytes 99 (Mybpc3fl/fl ; Myh6-Cre) (Figure 1D). By selectively measuring Mybpc3 mRNA that contained 100 exons 3 to 5 from these two transgenic models, we could determine the cardiomyocyte versus 101 non-cardiomyocyte source of Mybpc3 mRNA. We discovered that left ventricle Mybpc3 mRNA 102 expression was significantly reduced when cardiomyocyte Mybpc3 mRNA was eliminated 103 (Figure 1E). Likewise, lung and blood Mybpc3 mRNA was also significantly reduced when 104 cardiomyocyte Mybpc3 mRNA was eliminated (Figure 1F-G). Since our blood samples were 105 obtained by direct LV puncture, we also confirmed that Mybpc3 mRNA was detected in whole 106 blood obtained directly from the aorta ( Figure S1B). Overall, t hese results show that 107 extracardiac Mybpc3 mRNA is derived primarily from cardiomyocytes. 108 Extracardiac Mybpc3 mRNA does not lead to detectable MYBPC3 protein 109 Since we detected Mybpc3 mRNA in extracardiac tissues we wanted to determine if this 110 Mybpc3 mRNA leads to detectable MYBPC3 protein in extracardiac organs . We readily 111 detected MYBPC3 protein in heart left ventricle tissue lysate using two different primary 112 antibodies and two independent imaging methods (Figure 2A). In contrast to the left ventricle, 113 we were unable to detect MYBPC3 protein in extracardiac organ lysates such as lung, liver, 114 brain, kidney and skeletal muscle using these same methods ( Figure 2A-C). In addition, we 115 were unable to detect MYBPC3 protein in whole blood samples ( Figure 2D). Overall, these 116

show that extracardiac Mybpc3 mRNA does not lead to detectable MYBPC3 protein in 117 non-cardiac organs. 118 Non-cardiomyocyte cells of the myocardium do not express MYBPC3 protein 119 Since myocardial tissue was identified as the primary source of MYBPC3 mRNA and protein, 120 we next wanted to determine if non-cardiomyocyte cells had detectable MYBPC3 protein. We 121 again utilized our transgenic model that eliminated Mybpc3 exon 3-5 expression in all cells 122 (Mybpc3-/-) or specifically in cardiomyocytes (Mybpc3 fl/fl ; Myh6 -Cre). First, we used 123 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint immunohistochemistry and found that cardiomyocyte specific elimination of Mybpc3 led to no 124 detectable myocardial MYBPC3 protein (Figure 3A-B). Next, we performed proximity ligation 125 assays to quantify the in -situ MYBPC3 protein expression in cardiomyocytes and non -126 cardiomyocyte cell populations. Similar to the IHC experiment, cardiomyocytes had MYBPC3 127 protein complexes, but non -cardiomyocytes did not (Figure 3C-D, S 2A). Likewise, human 128 myocardial tissue cardiomyocytes expressed MYBPC3 protein , but non-cardiomyocytes did 129 not have detectable MYBPC3 protein expression (Figure 3E-F). To confirm the in situ data, we 130 also performed immunoblots on left ventricle tissue lysate from mice with cardiomyocyte 131 specific deletion of Mybpc3 and did not detect any residual MYBPC3 protein (Figure 3G-H, 132 S2B). 133 We then wanted to determine if human and murine cell lines expressed MYBPC3 protein since 134 it has been reported that some non -cardiomyocyte cell lines express sarcomere proteins .12 135 First, we compared human induced pluripotent stem cells (hiPSC) before and after 136 differentiation into cardiomyocytes (hiPSC -CM). Undifferentiated hiPSC lacked detectable 137 MYBPC3 or sarcomeric α-actinin protein, while hiPSC differentiated into cardiomyocytes had 138 detectable MYBPC3 and sarcomeric α-actinin protein (Figure 3I). Non-cardiomyocyte cell lines 139 from both humans and mice had no detectable MYBPC3 protein ( Figure 3 I). Overall, the 140

of these experiments show that cardiomyocytes are the cell source for MYBPC3 protein 141 in the left ventricle. 142 Cardiomyocyte versus germline Mybpc3 deletion causes similar pathologic remodeling 143 of the left ventricle 144 We then utilized our murine models to evaluate the impact of cardiomyocyte versus non -145 cardiomyocyte MYBPC3 protein expression on the development of cardiomyopathy. We found 146 that left ventricular wall thickness and dilation were similar in mice with germline deletion of 147 Mybpc3 versus cardiomyocyte specific deletion of Mybpc3 (Figure 4A-C). In addition, t here 148 were n o significant differences detected between male and female mice (Figure S3A-F). 149 Similar to the echocardiography results, heart mass (Figure 4D) and cardiomyocyte 150 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint hypertrophy (Figure 4E+F) were similar between germline versus cardiomyocyte specific 151 deletion of Mybpc3. In addition, we found similar levels of left ventricular fibrosis in mice with 152 either germline or cardiomyocyte specific deletion of Mybpc3 (Figure 4G-H). These results 153 show that selective elimination of MYBPC3 protein in cardiomyocytes leads to similar 154 pathologic remodeling of the left ventricle compared to elimination of MYBPC3 protein in all 155 murine cells. 156 Cardiomyocyte versus germline Mybpc3 deletion leads to similar abnormalities in left 157 ventricular systolic and diastolic function 158 Next, we evaluated if cardiomyocyte specific versus germline deletion of Mybpc3 led to 159 differences in left ventricular function. We found that left ventricular systolic function decreased 160 to a similar extent in both groups of mice in comparison to WT (Figure 5A). Likewise, left 161 ventricular diastolic function was impaired to a similar degree in mice with cardiomyocyte 162 Mybpc3 deletion versus germline Mybpc3 deletion (Figure 5B-D). Overall, these results show 163 that elimination of MYBPC3 protein in cardiomyocytes versus all cell types leads to similar 164 abnormalities in left ventricular systolic and diastolic function. 165

166 Mutations in the sarcomere gene, MYBPC3, are one of the most common causes of 167 hypertrophic cardiomyopathy in humans. Although MYBPC3 protein is an integral component 168 of the sarcomere of cardiomyocytes, both MYBPC3 mRNA and protein have been reported in 169 non-cardiomyocyte cell populations.10-12 Therefore, we utilized transgenic murine models that 170 enabled us to selectively eliminate cardiomyocyte Mybpc3 mRNA. This allowed us to 171 determine the cell source of extracardiac Mybpc3 mRNA and investigate whether non-172 cardiomyocyte gene expression influenced the development of MYBPC3 related 173 cardiomyopathy. We found that Mybpc3 mRNA was present in multiple extracardiac organs, 174 but cardiomyocytes were the primary source of this extracardiac Mybpc3 mRNA. In addition, 175 we found that despite the presence of extracardiac Mybpc3 mRNA, there was no detectable 176 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint MYBPC3 protein in extracardiac organs. Likewise, within the heart, there was no detectable 177 MYBPC3 protein in non-cardiomyocyte cell populations . Importantly, cardiomyocyte specific 178 elimination of MYBPC3 protein led to a similar effect on cardiac structure and function 179 compared to elimination of MYBPC3 in all cell types. Taken together, this study shows that 180 cardiomyocytes are the primary source of extracardiac Mybpc3 mRNA but this extracardiac 181 gene expression does not lead to detectable MYBPC3 protein or impact the development of 182 cardiomyopathy related to MYBPC3 deficiency. 183 Extracardiac MYBPC3 mRNA has been detected in whole blood samples in human patients 184 with HCM and has been utilized to determine the impact of MYBPC3 mutations on mRNA 185 splicing.17,18 We found that both humans and mice had detectable MYBPC3 mRNA in multiple 186 extracardiac organs. However, it was unclear what the cell source was for this extracardiac 187 MYBPC3 mRNA. We utilized o ur transgenic murine models to determine that extracardiac 188 Mybpc3 mRNA is primarily derived from cardiomyocytes since the elimination of cardiomyocyte 189 Mybpc3 mRNA expression caused a significant decrease in extracardiac Mybpc3 mRNA 190 levels. This suggests that cardiomyocytes can secrete Mybpc3 mRNA or passively release it 191 through cardiomyocyte rupture. Interestingly, it was found that blood levels of MYBPC3 RNA 192 increased in patients after ST -elevation myocardial infarction suggesting that cardiomyocyte 193 death may be one mechanism through which MYBPC3 mRNA is released into the blood. 19 194 Likewise, cardiomyocyte derived exosomes have been shown to contain both mRNA and 195 DNA.20 The mechanism s controlling MYBPC3 mRNA release and the biological role of 196 circulating MYBPC3 will need further investigation. 197 Interestingly, we detected residual low level s of Mybpc3 mRNA in lung tissue after 198 cardiomyocyte Mybpc3 mRNA expression was eliminated . The cell source of this non -199 cardiomyocyte Mybpc3 mRNA remains unclear but low levels of MYBPC3 mRNA have been 200 detected in human macrophages and neutrophils. 21,22 Likewise, it was previously shown that 201 Epstein-Barr virus immortalized lymphocytes can transcribe sarcomere genes such as myosin 202 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint heavy chain 7 (MYH7).23,24 Importantly, despite detecting Mybpc3 mRNA in lung tissue, we did 203 not detect MYBPC3 protein in lung tissue using multiple different MYBPC3 antibodies. 204 It was previously reported that MYBPC3 protein can be detected in myocardial derived 205 fibroblasts and NIH -3T3 cells .12 I n our transgenic models that had cardiomyocyte specific 206 deletion of Mybpc3, we were unable to detect residual MYBPC3 protein using in situ 207 immunofluorescence, proximity ligation assays of myocardial tissue sections or 208 immunoblotting of myocardial tissue lysate . Importantly, myocardial tissue samples from 209 animals with cardiomyocyte deletion of Mybpc3, had similar levels of myocardial fibrosis 210 compared to animals with germline deletion of Mybpc3 in all cells. These results suggest that 211 there were similar levels of pathologic fibroblast activation between the two models. Likewise, 212 we were unable to detect MYBPC3 protein in NIH -3T3 cells cultured under standard culture 213 conditions. Overall, our results show that non -cardiomyocyte cell populations of the murine 214 myocardium do not readily express MYBPC3 protein. 215 We found that cardiomyocyte specific deletion of Mybpc3 led to similar changes in cardiac 216 structure and function compared to germline deletion of Mybpc3 in all cells. This suggests that 217 the small amount of residual Mybpc3 mRNA expression in non-cardiomyocyte cell populations 218 has no discernable impact on cardiomyopathy development and progression in preclinical 219 murine models . These findings are particularly important because viral vector- based 220 approaches under development to treat human cardiomyopathies related to MYBPC3 protein 221 deficiency selectively target cardiomyocytes but not other cell populations.15,16 222

223 Taken together, this study shows that cardiomyocytes are the primary source of extracardiac 224 Mybpc3 mRNA and MYBPC3 protein is localized to the cardiomyocyte cell population. 225 Importantly, the expression of Mybpc3 mRNA in non-cardiomyocyte cell populations has no 226 discernible effect on the pathogenesis of cardiomyopathy resulting from MYBPC3 protein 227 deficiency. Therefore, selectively increasing cardiomyocyte MYBPC3 protein levels should be 228 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint the most efficient approach to mitigate the development of cardiomyopath ies caused by 229 MYBPC3 deficiency. 230

231 Ethics approval 232 All the mice that were used in this study were housed in animal facility accredited by the 233 American Association for the Accreditation of Laboratory Animal Care (AAALAC). The animal 234 experiments were conducted in accordance with the practices defined in the Guide for the Care 235 and Use of Laboratory Animals which were approved and overseen by the University of 236 Pittsburgh Institutional Animal Care and Use Committee (IACUC). 237 Mouse models 238 To determine the impact of loss of MYBPC3 protein in all cells, we utilized a murine model that 239 has a germline deletion of Mybpc3 exons 3 to 5 (Mybpc3 -/-).25 To achieve specific deletion of 240 Mybpc3 exons 3 to 5 in cardiomyocytes we crossed a Mybpc3fl/fl line with the cardiomyocyte 241 specific Myh6-Cre line (Jackson Labs, 011038). The generation of the Mybpc3-/- and Mybpc3fl/fl 242 lines were previously described.25 243 Human tissue analysis 244 Human control (unused donor) myocardial tissue samples were obtained in a deidentified 245 manner from an institutional review board-approved tissue biorepository. 246 Cell lines and culture 247 The human induced pluripotent stem cell s (hiPSCs) were obtained from the Standford SCBI 248 BioBank (SCVI274) and differentiated to hiPSC -derived cardiomyocytes (hiPSC -CMs) as 249 previously described.26 Briefly, hiPSCs were maintained in essential 8 medium ( A1517001, 250 Gibco). Once cells were confluent (~80%), 10 μM CHIR99021 (Selleckchem) was added in 251 RPMI 1640 media with B27 minus insulin (Gibco) for 48 hours. The media was then changed 252 to 5 μM IWR-1 (Sigma) in RPMI 1640 with B27 minus insulin for 48 hours. Cells were then 253 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint maintained with RPMI 1640 with B27 minus insulin every other day until differentiated cells 254 started to beat between 10 to 15 days post-differentiation. After 15 days of differentiation, iPSC-255 CMs were selected by RPMI 1640 without glucose supplemented with sodium L -lactate 256 (Sigma) every other day until day 19 post-differentiation. The cells were then given RPMI 1640 257 with glucose and insulin transferrin selenite (ITS) plus media supplement (AR014; R&D 258 systems) every other day. CMs were maintained with 5% CO2 at 37°C in a humidified incubator, 259 and 30 days post -differentiated matured cells were used for the experiment. The NIH-3T3 260 (mouse embryonic fibroblast cell line), HeLa (human cervical carcinoma epithelial cell line) and 261 HEK293T (human kidney epithelial cell line) cell lines were cultured separately in DMEM media 262 (GIBCO) supplement with 10% fetal bovine serum (FBS) with 5% CO2 at 37°C in humidified 263 incubator. 264 Echocardiography 265 To assess the mouse cardiac systolic function and structure we performed transthoracic 266 echocardiography using a Vevo 3100 (VisualSonics, Inc) without anesthesia. The 267 interventricular septal thickness at end-diastole (IVSd), the left ventricular posterior wall at end-268 diastole (LVPWd), the left ventricular internal dimensions at end -systole (LVIDs) and at end-269 diastole (LVIDd) were acquired from M-mode short axis images. The left ventricular fractional 270 shortening (FS) was obtained with the following formula (LVIDd – LVIDs)/LVIDd. 271 The mouse diastolic function was measured with B-mode long axis four chamber view under 272 continuous isoflurane anesthesia. The left ventricular relaxation peak velocity in early diastole 273 (E) and atrial contraction peak velocity in late diastole (A) was measured by mitral valve 274 doppler flow. Early (E’) and late (A’) diastolic mitral annular tissue velocity was assessed by 275 tissue Doppler image analysis. Finally, the Isovolumic relaxation time (IVRT) was obtained by 276 measuring the period between the aortic valve closure and the mitral valve opening. 277 Euthanasia and heart mass assessment 278 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Mice were sedated with 5% isoflurane, and once unresponsive to toe pinch, body weight was 279 measured. Before excising the heart, 500μL of blood was collected from the left ventricle (LV) 280 or aortic puncture. The heart was then removed and heart weight was recorded. Mouse left 281 ventricle tissue was isolated and then snap -frozen in liquid nitrogen for RNA and protein 282 analysis. Full hearts were embedded in OCT for histology and immunohistochemistry staining. 283 All the tissue samples were banked in -80°C freezer prior to being processed in future 284 experiments. Mouse lower extremities were then amputated at the mid femur level and boiled 285 to remove excess tissues and isolate the tibia. Digital calipers were used to measure the tibia 286 length. Heart weight to tibia length (mm) ratios were then calculated. 287 Histology 288 To determine cardiac fibrosis deposition, 5-μm thick sections were collected by cryo-sectioning 289 the OCT-embedded heart. The harvested sections were then fixed for 5 minutes at -20°C in 290 Acetone, then air -dried for 20 minutes at room temperature prior to being rehydrated in 1X 291 PBS wash for 1 minute. Sections were then stained at room temperature for 1 hour with Sirius 292 Red/Fast green solution (Chondrex 90461) before being washed in distilled water. 293 Sections were then imaged with a Zeiss Axioplan microscope to get bright-field image at x40 294 magnification. 10 pictures were taken per tissue sample. To determine the percentage of 295 fibrotic area these pictures were analyzed with ImageJ software. To assess the fibrotic area, 296 tissue stained with Sirius red was measured. Additionally, the myocardial area was determined 297 by measuring the red and green stained tissue. The percentage of fibrotic area was then 298 obtained by establishing a ratio of the fibrotic area per myocardial area for the tissue section 299 of interest. 300 Immunofluorescence and Wheat Germ Agglutinin Staining 301 Hearts were embedded in OCT prior to being sectioned at 5-μm on a cryostat (Thermo Fisher 302 Scientific) and then harvested on Superfrost Plus Gold microscope slides (Fisher Scientific). 303 Sections were fixed for 15 minutes with 4% paraformaldehyde then permeabilized with 0.2% 304 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Triton X-100 for 15 minutes with subsequent PBS washes. For the MYBPC3-WGA co-staining, 305 an additional blocking step was performed by incubating the tissue sections with 0.1mg/mL 306 Fab fragment goat anti -mouse IgG (Jackson Immuno Research, 115 -07-003) at room 307 temperature for 1 hour. Sections were then incubated for 1 hour with blocking buffer (1% BSA 308 in 1X PBS). Sections were then incubated overnight at 4°C with MYBPC3 antibody (Santa 309 Cruz Biotechnology, sc-137180, E-7). The following day tissue sections were washed and 310 incubated for 1 hour at room temperature with fluorescent secondary antibody goat anti-mouse 311 Alexa fluor 594 (Thermo Fisher Scientific, A-11032), goat anti-rabbit Alexa fluor 594 (Thermo 312 Fisher Scientific, A-11005) and then washed in 1X PBS. Sections were then stained with wheat 313 germ agglutinin (WGA) (Thermo Fisher Scientific, W6748) 1:200 at room temperature for 1 314 hour with subsequent washes in 1X PBS. Sections were then counterstained and mounted 315 with Prolong Gold Antifade with 4’6-diamidino-2-phenylindole (DAPI). Slides were then imaged 316 under wide-field fluorescent microscope (Zeiss) at x40 magnification. MYBPC3 fluorescence 317 was determined by using the following equation: corrected MYBPC3 fluorescence = integrated 318 density – (area of cardiomyocyte x mean of fluorescence of background readings), where 319 integrated density is fluorescence intensity of the defined region of interest, are a of 320 cardiomyocyte is the size of the defined region of interest and mean of fluorescence 321

is the average intensity of 3 background regions of interest. 322 Proximity ligation assay 323 In situ Proximity ligation assay (PLA) was performed as previously described. 27 Mouse and 324 human frozen heart tissue sections were cryosectioned 5μm thick. They were then fixed with 325 4% PFA for 15 minutes and washed with 1X PBS. The tissue sections were permeabilized with 326 0.2% Triton X-100 with subsequent PBS washes. Sections were then blocked using Duolink 327 blocking buffer (Sigma) for 1 hour at RT and incubated with primary antibodies mouse 328 MYBPC3 (Santa Cruz Biotechnology, sc-137180, E-7) 1:1000, and rabbit MYBPC3 (Invitrogen, 329 703574, 19H1L3) 1:1000 diluted in Duolink antibody diluent , overnight at 4°C. The sections 330 were then incubated with secondary antibodies labeled with PLA probes (anti-mouse MINUS 331 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint and anti-rabbit PLUS) diluted 1:5 in 1X Duolink Antibody Diluent buffer at 37°C for 1 hour. The 332 ligation step was performed by adding ligase diluted in ligation buffer to the heart tissue section 333 at 37°C for 30 minutes . The amplification step was then performed by adding polymerase 334 diluted in ampli fication buffer in the dark at 37°C for 100 minutes. Secondary antibody 335 incubation, ligation, and amplification steps were all completed in a humidity chamber. After 336 final PBS washes, the heart tissue sections were incubated for 1 hour with blocking buffer (1% 337 BSA in 1X PBS) and incubated with sarcomeric α-actinin antibody (Abcam, Ab9465) (1:1000) 338 overnight at 4°C. After overnight incubation, the tissue sections were then washed and 339 incubated for 1 hour at room temperature with fluorescent secondary antibody goat anti-mouse 340 Alexa fluor 488 (Thermo Fisher Scientific, A-10667) followed by subsequent PBS washes. The 341 heart tissue sections were then mounted with Prolong Gold Antifade with 4’6 -diamidino-2-342 phenylindole (DAPI) (Thermo Fisher). 343 Slides were imaged under a wide-field fluorescent microscope (Zeiss) at 40 x magnification. 344 MYBPC3 complexes were estimated by thresholding the MYBPC3 PLA signal to measure the 345 amount of MYBPC3 complexes located in the myocardium and the interstitial space. The 346 average area, in pixels, of 25 individual PLA dots was measured across three randomly 347 selected areas of the myocardium. In ImageJ, images were converted to RGB stack format. 348 The percentage of red fluorescence in each image was thresholded using the ImageJ threshold 349 function using an upper limit of 255, and a lower limit that was adjusted for each image to fully 350 capture all of the red fluorescence. The percentage of red fluorescence was then multiplied by 351 the total number of pixels in the image to calculate the total red pixels per image. This value 352 was divided by the average PLA dot area to estimate the amount of MYBPC3 complexes per 353 image. Non-cardiomyocyte MYBPC3 complexes were counted manually and subtracted from 354 the total number of complexes to obtain the number of cardiomyocyte specific MYBPC3 355 complexes per image. The c ardiomyocyte area in µm 2 was measured by thresholding the 356 sarcomeric α-actinin stained area (green channel) to obtain a percentage of green 357 fluorescence in each image, which was then multiplied by the total image area. The non -358 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint cardiomyocyte area was calculated by subtracting the cardiomyocyte area from the total image 359 area. Cardiomyocyte MYBPC3 complexes per 100 µm 2 were determined by dividing the 360 number of cardiomyocyte MYBPC3 complexes by the cardiomyocyte area (µm 2) and then 361 multiplying by 100. Non -cardiomyocyte MYBPC3 complexes per 100 µm 2 were obtained by 362 dividing the non -cardiomyocyte MYBPC3 complexes by the non -cardiomyocyte area and 363 multiplying by 100. 364 Human bulk tissue RNA-sequencing 365 MYBPC3 read counts for heart left ventricle, whole blood, lung, liver, kidney cortex, brain frontal 366 cortex and skeletal muscle were obtained from the GTEx Portal on 08/05/2024 and dbGaP 367 accession number phs000424.v 8.p2 on 08/05/2024. The MYBPC3 RNA expression were 368 normalized as a Log10(read counts). 369 qRT-PCR 370 50mg of mouse left ventricle tissue was homogenized in 500μL of TRIzol®, while 500μL of 371 mouse blood (harvested from LV and aorta puncture) were mixed with 500μL of TRIzol®. Then 372 RNA extraction was performed following the protocol provided by the manufacturer (Direct -373 zolTM RNA Miniprep, Zymo Research). Reverse transcription was performed following the 374 manufacturer directions (Verso cDNA synthesis kit, Thermo Fisher Scientific). cDNA synthesis 375 was performed using random hexamer primers except when specifically detailed as oligo dT 376 primers. To assess gene expression, qPCR was performed with the Syber Green Master Mix 377 (Applied Biosystem, A25742), Mybpc3 specific primers were designed to bind to the splice 378 junctions of exons 2+3 and 3+4 (Table S1). cDNA samples were not diluted prior to running a 379 40 cycle qPCR reaction. Plate was set up using Quant -Studio-5 Real Time PCR System 380 (Applied Biosystem). Each sample was run in duplicates or triplicates, and the Ct value was 381 normalized using the housekeeping gene Rpl32. The 2-∆∆Ct method was used to calculate the 382 fold change in Mybpc3 mRNA expression relative to indicated group. Since the maximum cycle 383 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint number was 40 for the qPCR, a Ct value of 40 was assigned to samples that were reported as 384 undetected. 385 Protein Electrophoresis and Western Blot 386 30mg of mouse left ventricle tissue were homogenized in 300μL ice-cold RIPA buffer (Sigma) 387 supplemented with protease/phosphatase inhibitors (Thermo Fisher Scientific, 78441). 50μg 388 of proteins from mouse left ventricle and 30 μg of proteins from cell lines were separated on 389 SDS-PAGE using 10% Tris -Glycine gel (Bio -Rad, 4561093, 5671033 ) and transferred to a 390 0.45-μm low -fluorescence PVDF membrane (Bio-Rad) at 4°C for 1 hour at 100V. Total protein 391 was imaged on the membrane with the Revert TM 700 total protein stain kit (LI-COR, 926-392 11016). Membrane was then blocked with 5% BSA or non-fat dry milk in TBS/Tween20 (0.1% 393 v/v) at room temperature for 1 hour. Primary antibody was incubated at 4°C overnight with 394 MYBPC3 (Santa Cruz Biotechnology, sc-137180, E-7), (Invitrogen, 703574, 19H1L3), or β-395 actin (Cell Signaling Technology, 8457) in 1:500. The following day membranes were washed 396 with TBST and incubated with secondary antibody goat anti -mouse HRP ( Cell Signaling 397 Technology, 7076S), or goat anti -rabbit HRP ( Cell Signaling Technology , 7074S) at room 398 temperature for 1 hour with subsequent washes and then imaged with Chemi Doc apparatus 399 (Bio-Rad) using Clarity ECL Substrate (Bio-Rad, 1705061). For LI-COR imaging system, goat 400 anti-mouse (IRDye 680 LT, 926-68020) and goat anti-rabbit (IRDye 800 CW, 926-32211) were 401 used, and membranes were imaged with Odyssey CLx imaging system (LI-COR). 402 Statistical analysis 403 All experimental data are displayed as mean ± SEM. The normal (Gaussian) distribution of the 404 experimental data set was tested with the Shapiro -Wilk normality test. If the data set were 405 normally distributed, statistical significance between two experimental groups was tested using 406 two-tailed unpaired Student’s t-test. However, if the data set failed to pass the F test to compare 407 variances, therefore, a Welch’s t-test was performed. For the data set including more than two 408 experimental group s a one -way ANOVA with a Tukey’s multiple comparisons test was 409 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint performed to test the statistical significance. However, if the data set failed to pass the F test 410 to compare variances, therefore, a Brown -Forsythe and Welch ANOVA with Dunnetts’s T3 411 multiple group comparisons test was performed. For data sets that were not normally 412 distributed, Mann -Whitney U test was used to test statistical significance between two 413 experimental groups. However, Kruskal-Wallis test with Dunn’s post hoc test for multiple group 414 comparison for data sets including more than two experimental groups. All statistical analysis 415 was performed with Prism 10 software (GraphPad). Statistical significance was considered a 416 p-value less than 0.05. 417

418 We would like to acknowledge Dr. Sruti Shiva (Heart, Lung, Blood Vascular Institute, University 419 of Pittsburgh) and Dr. Yael Nechemia-Arbely (Hillman cancer center, University of Pittsburgh) 420 for providing us with NIH-3T3 and HeLa cells respectively. 421 Author contributions 422 N.G.C and J.R.B designed the research study. N.G.C ., K.P.L., S.T.M, J.H.K., K.S., S.P., and 423 J.R.B. conducted experiments and data analysis. N.G.C., K.P.L., S.T.M, J.H.K., K.S., S.P., and 424 J.R.B. prepared and edited the manuscript. 425 Source of Funding 426 This work was supported by grants from the National Institutes of Health (HL136824, 427 HL160890, HL167955, and HL169784 to J.R.B.) 428 Disclosures 429 None. 430 Supplementary Data 431 Figure S1, S2, S3 and Table S1 432 433 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint

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TargeMng Cardiomyocyte PCNA and POLD1 Prevents Pathologic 524 Myocardial Hypertrophy. Circ Res . 2025;137:1160 -1181. doi: 525 10.1161/CIRCRESAHA.124.325647 526 527 528 529 530 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure Legends 531 Figure 1: Cardiomyocytes are the primary source of extracardiac Mybpc3 mRNA 532 (A) Human bulk t issue RNA -sequencing for MYBPC3 expression from Genotype -Tissue 533 Expression (GTEx) database. MYBPC3 mRNA expression is displayed as Log10(read counts) 534 for cardiac left ventricle (n=432) , whole blood (n=755), lung (n=578), liver (n=226), kidney 535 (n=85), brain (n=255) and skeletal muscle (n=803). (B) Quantification of Mybpc3 expression 536 from Mybpc3-/- and WT heart left ventricle (n=6-12), lung (n=5-11), whole blood (n=6-12), liver 537 (n=6) and skeletal muscle (n=5) at postnatal day (P) 90. The Mybpc3 gene expression was 538 normalized to the housekeeping gene Rpl32 and the cDNA was created using random 539 hexamer primers . Results are expressed as a fold change relative to Mybpc3 mRNA 540 expression in the Mybpc3-/- mouse tissue. (C-D) Schematic of the Mybpc3 germline knockout 541 (Mybpc3-/-) and cardiomyocyte specific deletion ( Mybpc3fl/fl ; Myh6-Cre). Schematic drawing 542 was performed with BioRender software. (E) Quantification of Mybpc3 expression from 543 Mybpc3-/- (n=5), WT (n=12) and Mybpc3fl/fl ; Myh6-Cre (n=12) heart left ventricle at P90. The 544 Mybpc3 gene expression was normalized to the housekeeping gene Rpl32. Results are 545 expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- hearts. (F) 546 Quantification of Mybpc3 expression from Mybpc3-/- (n=4), WT (n=11) and Mybpc3fl/fl ; Myh6-547 Cre (n=11) lung at P90. The Mybpc3 gene expression was normalized to the housekeeping 548 gene expression Rpl32. Results are expressed as a fold change relative to Mybpc3 mRNA 549 expression in the Mybpc3-/- lungs. (G) Quantification of Mybpc3 expression from, Mybpc3-/- 550 (n=6), WT (n=9) and Mybpc3fl/fl ; Myh6-Cre (n=10) blood at P90. The Mybpc3 gene expression 551 was normalized to the housekeeping gene expression Rpl32. Results are expressed as a fold 552 change relative to Mybpc3 mRNA expression in the Mybpc3-/- blood. All results are shown as 553 mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons for was 554 used for A, E, F and G. Mann Whitney U test was used for B to compare WT and Mybpc3-/- for 555 heart left ventricle, lung, whole blood, liver and skeletal muscle. 556 557 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 2: Extracardiac Mybpc3 mRNA does not lead to detectable MYBPC3 protein 558 (A) Immunoblot images of MYBPC3 protein expression from WT and Mybpc3-/- mice in heart 559 left ventricle, lung, liver, kidney, brain and skeletal muscle from mouse tissue at post-natal day 560 180 (P180). The total protein stain was used as the loading control. The immunoblots used 561 MYBPC3 antibodies 19H1L3 (Invitrogen, 703574) or E-7 (Santa Cruz Biotechnology, sc -562 137180) and were imaged with ChemiDoc (exposure time detailed) or LI-COR imaging 563 systems. (B) Representative immunoblot images of MYBPC3 protein expression from WT mice 564 in heart left ventricle, lung, liver, kidney, brain and skeletal muscle from mouse tissue at P180. 565 The total protein stain was used as the loading control . The immunoblot used MYBPC3 566 antibody 19H1L3 (Invitrogen, 703574 ) and was imaged with ChemiDoc (exposure time 567 detailed). (C) MYBPC3 protein quantification from WT (n=6) left ventricle, lung, liver, kidney, 568 brain and skeletal muscle mouse tissue at P180. (D) Immunoblot images of MYBPC3 protein 569 expression from WT and Mybpc3-/- mice in whole blood from aortic puncture using a MYBPC3 570 antibody 19H1L3 (Invitrogen, 703574) and imaged with ChemiDoc (exposure time detailed). 571 The housekeeping protein β-actin was used as the loading control . All results are shown as 572 mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons was used 573 for C. 574 Figure 3: Non-cardiomyocyte cells of the myocardium do not express MYBPC3 protein 575 (A) Representative images of immunofluorescence staining of WT, Mybpc3-/- and Mybpc3fl/fl ; 576 Myh6-Cre left ventricle mouse tissue at postnatal day 90 (P90). MYBPC3 - red, wheat germ 577 agglutinin (WGA) - green, 4’6-diamidino-2-phenylindole (DAPI) - blue Scale bars, 25μm. (B) 578 MYBPC3 fluorescence intensity quantification from WT (n=6), Mybpc3-/- (n=5) and Mybpc3fl/fl ; 579 Myh6-Cre (n=6) at P90. Minimum of 100 cardiomyocytes/sample. (C) Representative images 580 of a in situ proximity ligation assay for MYBPC3 (red) counter stained with sarcomeric α actinin 581 (green) and DAPI (blue) in WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre left ventricle tissue at 582 P180. Scale bars, 25μm. (D) In situ proximity ligation assay to quantify MYBPC3 protein 583 complexes (per 100μm²) in cardiomyocytes versus non-cardiomyocytes in WT, Mybpc3-/- and 584 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Mybpc3fl/fl ; Myh6-Cre (n=6/group) left ventricle tissue at P180 . (E) Representative images of 585 an in situ proximity ligation assay for MYBPC3 (red) counter stained with sarcomeric α actinin 586 (green) and DAPI (blue) in human control (n=4) left ventricle . Scale bars, 25μm. (F) 587 Quantification of MYBPC3 complexes (complexes per 100μm²) in cardiomyocytes versus non-588 cardiomyocytes in human control (n=4) left ventricle. (G) Immunoblot images of MYBPC3 and 589 Sarcomeric α actinin protein expression from WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre mice 590 left ventric le tissue lysate at P180 . The immunoblot s used MYBPC3 antibodies 19H1L3 591 (Invitrogen, 703574) or E-7 (Santa Cruz Biotechnology, sc -137180) and were imaged with 592 ChemiDoc (exposure time detailed) or LI-COR imaging systems. β-actin was used as a loading 593 control. (H) MYBPC3 protein quantification from WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6 -Cre 594 (n=3/group) left ventricl e mouse tissue at P180 . Quantification was performed using t he 595 immunoblot image from antibody 19H1L3 with 1 minute exposure time on ChemiDoc. (I) 596 Immunoblot images of MYBPC3 protein expression from human induced pluripotent stem cells 597 (hiPSC), human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM), HeLa, 598 HEK293T and NIH-3T3 cell lines. The total protein stain was used as the loading control. All 599

are shown as mean±SEM. Kruskal -Wallis test with Dunn’s post hoc test for multiple 600 comparisons for was used for B and H. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 601 multiple comparisons test was used for D. Unpaired t test with Welch’s correction was used for 602 F. 603 Figure 4: Cardiomyocyte versus germline Mybpc3 deletion causes similar pathologic 604 remodeling of the left ventricle 605 Transthoracic echocardiography was performed to measure (A) interventricular septal 606 thickness at end-diastole (IVSd), (B) left ventricular posterior wall at end-diastole (LVPWd), (C) 607 and left ventricular internal dimension at end -diastole (LVIDd) from WT (n=11-12), Mybpc3-/- 608 (n=13-14) and Mybpc3fl/fl ; Myh6-Cre (n=15) at postnatal day 25 ( P25) and P90. (D) Heart 609 weight (HW) to tibia length (TL) ratio from WT (n=6), Mybpc3-/- (n=7) and Mybpc3fl/fl ; Myh6-610 Cre (n=7) at P90. (E) Representative images of wheat germ agglutinin (WGA) (green) and 4’6-611 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint diamidino-2-phenylindole (DAPI) (blue) fluorescence co -staining from WT (n=6), Mybpc3-/- 612 (n=5) and Mybpc3fl/fl ; Myh6-Cre (n=6) at P90. Scale bars, 25 μm. (F) Cardiomyocyte cross-613 sectional area quantification from WGA staining. Minimum of 100 cardiomyocytes/sample. (G) 614 Representative images of Sirius Red/Fast Green staining from WT (n=5), Mybpc3-/- (n=6) and 615 Mybpc3fl/fl ; Myh6-Cre (n=6) at P90. Scale bars, 25 μm. (H) Myocardial fibrosis quantification 616 from Sirius Red/Fast Green staining. All results are shown as mean±SEM. Kruskal-Wallis test 617 with Dunn’s post hoc test for multiple comparisons was used for A, B and D. One-way ANOVA 618 with Tukey’s multiple comparisons test was used for F and H. Brown-Forsythe and Welch 619 ANOVA with Dunnett’s T3 multiple comparisons test was used for C. 620 Figure 5: Cardiomyocyte versus germline Mybpc3 deletion leads to similar 621 abnormalities in left ventricular systolic and diastolic function 622 Transthoracic echocardiography was performed to measure (A) fractional shortening (FS) from 623 WT (n=11-12), Mybpc3-/- (n=13-14) and Mybpc3fl/fl ; Myh6-Cre (n=15) at postnatal day 25 (P25) 624 and P90. Transthoracic echocardiography was performed to measure (B) isovolumic relaxation 625 time (IVRT), (C) mitral valve early to late filling velocity ratio (E/A) and (D) early transmitral 626 valve flow velocity to early mitral annulus tissue velocity ratio (E/e’) from WT (n=7), Mybpc3-/- 627 (n=7) and Mybpc3fl/fl ; Myh6-Cre (n=8) at P90. All results are shown as mean±SEM. Kruskal-628 Wallis test with Dunn’s post hoc test for multiple comparisons for was used for A. One-way 629 ANOVA with Tukey’s multiple comparisons test was used for B and C. Brown-Forsythe and 630 Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for D. 631 Supplementary Figure Legend 632 Figure S1 633 (A) Quantification of Mybpc3 expression from Mybpc3-/- and WT heart left ventricle (n=6-13), 634 lung (n=6-13) and whole blood (n=5-24) at P90. The Mybpc3 gene expression was normalized 635 to the housekeeping gene Rpl32 and the cDNA was created using oligo(dT) primers . (B) 636 Quantification of Mybpc3 mRNA expression from Mybpc3-/- and WT whole blood from aort ic 637 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint puncture (n=5-6), at postnatal day 90 (P90). The Mybpc3 gene expression was normalized to 638 the housekeeping gene Rpl32. Results are expressed as a fold change relative to Mybpc3 639 mRNA expression in the Mybpc3-/- mouse tissue. All results are shown as mean±SEM. Mann-640 Whitney U test was utilized for A + B. 641 Figure S2 642 (A) In situ proximity ligation assay (PLA) quantification for MYBPC3 complexes per field in left 643 ventricular tissue from WT (n=6), Mybpc3-/- (n=6), and Mybpc3fl/fl;Myh6Cre (n=6) at P180. (B) 644 Immunoblot images of MYBPC3 protein from WT and Mybpc3fl/fl ; Myh6-Cre left ventricular 645 tissue using different concentrations of total protein lysate (100μg, 200μg and 300μg). The total 646 protein stain was used as the loading control. All results are shown as mean±SEM. Brown-647 Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for A. 648 Figure S3 649 Transthoracic echocardiography was performed to measure (A) interventricular septal 650 thickness at end-diastole (IVSd), (B) left ventricular posterior wall at end-diastole (LVPWd), (C) 651 and left ventricular internal dimensions at end -diastole (LVIDd) from Mybpc3-/- (male n=5; 652 female n=8) and Mybpc3fl/fl ; Myh6-Cre (male n=7; female n=8) at P90. All results are shown 653 as mean±SEM. Student’s Welch’s t test was used for A and B. Mann Whitney U test was for 654 C. Unpaired Student’ t test was used for D, E and F. 655 Supplementary Table Legend 656 Table S1 657 List of oligonucleotide primer sequences used for qRT-PCR. 658 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 1: Cardiomyocytes are the primary source of extracardiac Mybpc3 mRNA (A) Human bulk tissue RNA-sequencing for MYBPC3 expression from Genotype-Tissue Expression (GTEx) database. MYBPC3 mRNA expression is displayed as Log10(read counts) for cardiac left ventricle (n=432), whole blood (n=755), lung (n=578), liver (n=226), kidney (n=85), brain (n=255) and skeletal muscle (n=803). (B) Quantification of Mybpc3 expression from Mybpc3-/- and WT heart left ventricle (n=6-12), lung (n=5-11), whole blood (n=6-12), liver (n=6) and skeletal muscle (n=5) at postnatal day (P) 90. The Mybpc3 gene expression was normalized to the housekeeping gene Rpl32 and the cDNA was created using random hexamer primers. Results are expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- mouse tissue. (C-D) Schematic of the Mybpc3 germline knockout (Mybpc3-/-) and cardiomyocyte specific deletion (Mybpc3fl/fl ; Myh6-Cre). Schematic drawing was performed with BioRender software. (E) Quantification of Mybpc3 expression from Mybpc3-/- (n=5), WT (n=12) and Mybpc3fl/fl ; Myh6-Cre (n=12) heart left ventricle at P90. The Mybpc3 gene expression was normalized to the housekeeping gene Rpl32. Results are expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- hearts. (F) Quantification of Mybpc3 expression from Mybpc3-/- (n=4), WT (n=11) and Mybpc3fl/fl ; Myh6-Cre (n=11) lung at P90. The Mybpc3 gene expression was normalized to the housekeeping gene expression Rpl32. Results are expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- lungs. (G) Quantification of Mybpc3 expression from, Mybpc3-/- (n=6), WT (n=9) and Mybpc3fl/fl ; Myh6-Cre (n=10) blood at P90. The Mybpc3 gene expression was normalized to the housekeeping gene expression Rpl32. Results are expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- blood. All results are shown as mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons for was used for A, E, F and G. Mann Whitney U test was used for B to compare WT and Mybpc3-/- for heart left ventricle, lung, whole blood, liver and skeletal muscle. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 2: Extracardiac Mybpc3 mRNA does not lead to detectable MYBPC3 protein (A) Immunoblot images of MYBPC3 protein expression from WT and Mybpc3-/- mice in heart left ventricle, lung, liver, kidney, brain and skeletal muscle from mouse tissue at post-natal day 180 (P180). The total protein stain was used as the loading control. The immunoblots used MYBPC3 antibodies 19H1L3 (Invitrogen, 703574) or E-7 (Santa Cruz Biotechnology, sc-137180) and were imaged with ChemiDoc (exposure time detailed) or LI-COR imaging systems. (B) Representative immunoblot images of MYBPC3 protein expression from WT mice in heart left ventricle, lung, liver, kidney, brain and skeletal muscle from mouse tissue at P180. The total protein stain was used as the loading control. The immunoblot used MYBPC3 antibody 19H1L3 (Invitrogen, 703574) and was imaged with ChemiDoc (exposure time detailed). (C) MYBPC3 protein quantification from WT (n=6) left ventricle, lung, liver, kidney, brain and skeletal muscle mouse tissue at P180. (D) Immunoblot images of MYBPC3 protein expression from WT and Mybpc3-/- mice in whole blood from aortic puncture using a MYBPC3 antibody 19H1L3 (Invitrogen, 703574) and imaged with ChemiDoc (exposure time detailed). The housekeeping protein β-actin was used as the loading control. All results are shown as mean±SEM. Kruskal- Wallis test with Dunn’s post hoc test for multiple comparisons was used for C. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 3: Non-cardiomyocyte cells of the myocardium do not express MYBPC3 protein (A) Representative images of immunofluorescence staining of WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre left ventricle mouse tissue at postnatal day 90 (P90). MYBPC3 - red, wheat germ agglutinin (WGA) - green, 4’6-diamidino-2- phenylindole (DAPI) - blue Scale bars, 25μm. (B) MYBPC3 fluorescence intensity quantification from WT (n=6), Mybpc3-/- (n=5) and Mybpc3fl/fl ; Myh6-Cre (n=6) at P90. Minimum of 100 cardiomyocytes/sample. (C) Representative images of a in situ proximity ligation assay for MYBPC3 (red) counter stained with sarcomeric α actinin (green) and DAPI (blue) in WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre left ventricle tissue at P180. Scale bars, 25μm. (D) In situ proximity ligation assay to quantify MYBPC3 protein complexes (per 100μm²) in cardiomyocytes versus non-cardiomyocytes in WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre (n=6/group) left ventricle tissue at P180. (E) Representative images of an in situ proximity ligation assay for MYBPC3 (red) counter stained with sarcomeric α actinin (green) and DAPI (blue) in human control (n=4) left ventricle. Scale bars, 25μm. (F) Quantification of MYBPC3 complexes (complexes per 100μm²) in cardiomyocytes versus non-cardiomyocytes in human control (n=4) left ventricle. (G) Immunoblot images of MYBPC3 and Sarcomeric α actinin protein expression from WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre mice left ventricle tissue lysate at P180. The immunoblots used MYBPC3 antibodies 19H1L3 (Invitrogen, 703574) or E-7 (Santa Cruz Biotechnology, sc-137180) and were imaged with ChemiDoc (exposure time detailed) or LI-COR imaging systems. β-actin was used as a loading control. (H) MYBPC3 protein quantification from WT, Mybpc3-/- and Mybpc3fl/fl ; Myh6-Cre (n=3/group) left ventricle mouse tissue at P180. Quantification was performed using the immunoblot image from antibody 19H1L3 with 1 minute exposure time on ChemiDoc. (I) Immunoblot images of MYBPC3 protein expression from human induced pluripotent stem cells (hiPSC), human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM), HeLa, HEK293T and NIH-3T3 cell lines. The total protein stain was used as the loading control. All results are shown as mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons for was used for B and H. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for D. Unpaired t test with Welch’s correction was used for F. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 4: Cardiomyocyte versus germline Mybpc3 deletion causes similar pathologic remodeling of the left ventricle Transthoracic echocardiography was performed to measure (A) interventricular septal thickness at end-diastole (IVSd), (B) left ventricular posterior wall at end-diastole (LVPWd), (C) and left ventricular internal dimension at end-diastole (LVIDd) from WT (n=11-12), Mybpc3-/- (n=13-14) and Mybpc3fl/fl ; Myh6-Cre (n=15) at postnatal day 25 (P25) and P90. (D) Heart weight (HW) to tibia length (TL) ratio from WT (n=6), Mybpc3-/- (n=7) and Mybpc3fl/fl ; Myh6-Cre (n=7) at P90. (E) Representative images of wheat germ agglutinin (WGA) (green) and 4’6-diamidino-2-phenylindole (DAPI) (blue) fluorescence co-staining from WT (n=6), Mybpc3-/- (n=5) and Mybpc3fl/fl ; Myh6-Cre (n=6) at P90. Scale bars, 25μm. (F) Cardiomyocyte cross-sectional area quantification from WGA staining. Minimum of 100 cardiomyocytes/sample. (G) Representative images of Sirius Red/Fast Green staining from WT (n=5), Mybpc3-/- (n=6) and Mybpc3fl/fl ; Myh6-Cre (n=6) at P90. Scale bars, 25μm. (H) Myocardial fibrosis quantification from Sirius Red/Fast Green staining. All results are shown as mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons was used for A, B and D. One-way ANOVA with Tukey’s multiple comparisons test was used for F and H. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for C. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure 5: Cardiomyocyte versus germline Mybpc3 deletion leads to similar abnormalities in left ventricular systolic and diastolic function Transthoracic echocardiography was performed to measure (A) fractional shortening (FS) from WT (n=11-12), Mybpc3-/- (n=13-14) and Mybpc3fl/fl ; Myh6-Cre (n=15) at postnatal day 25 (P25) and P90. Transthoracic echocardiography was performed to measure (B) isovolumic relaxation time (IVRT), (C) mitral valve early to late filling velocity ratio (E/A) and (D) early transmitral valve flow velocity to early mitral annulus tissue velocity ratio (E/e’) from WT (n=7), Mybpc3-/- (n=7) and Mybpc3fl/fl ; Myh6-Cre (n=8) at P90. All results are shown as mean±SEM. Kruskal-Wallis test with Dunn’s post hoc test for multiple comparisons for was used for A. One-way ANOVA with Tukey’s multiple comparisons test was used for B and C. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for D. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure S1 (A) Quantification of Mybpc3 expression from Mybpc3-/- and WT heart left ventricle (n=6-13), lung (n=6-13) and whole blood (n=5-24) at P90. The Mybpc3 gene expression was normalized to the housekeeping gene Rpl32 and the cDNA was created using oligo(dT) primers. (B) Quantification of Mybpc3 mRNA expression from Mybpc3-/- and WT whole blood from aortic puncture (n=5-6), at postnatal day 90 (P90). The Mybpc3 gene expression was normalized to the housekeeping gene Rpl32. Results are expressed as a fold change relative to Mybpc3 mRNA expression in the Mybpc3-/- mouse tissue. All results are shown as mean±SEM. Mann-Whitney U test was utilized for A + B. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure S2 (A) In situ proximity ligation assay (PLA) quantification for MYBPC3 complexes per field in left ventricular tissue from WT (n=6), Mybpc3-/- (n=6), and Mybpc3fl/fl;Myh6Cre (n=6) at P180. (B) Immunoblot images of MYBPC3 protein from WT and Mybpc3fl/fl ; Myh6-Cre left ventricular tissue using different concentrations of total protein lysate (100μg, 200μg and 300μg). The total protein stain was used as the loading control. All results are shown as mean±SEM. Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test was used for A. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Figure S3 Transthoracic echocardiography was performed to measure (A) interventricular septal thickness at end-diastole (IVSd), (B) left ventricular posterior wall at end-diastole (LVPWd), (C) and left ventricular internal dimensions at end-diastole (LVIDd) from Mybpc3-/- (male n=5; female n=8) and Mybpc3fl/fl ; Myh6-Cre (male n=7; female n=8) at P90. All results are shown as mean±SEM. Student’s Welch’s t test was used for A and B. Mann Whitney U test was for C. Unpaired Student’ t test was used for D, E and F. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint Table S1 List of oligonucleotide primer sequences used for qRT-PCR. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 23, 2026. ; https://doi.org/10.64898/2026.04.20.718297doi: bioRxiv preprint