Cardiomyocyte specific Bmal1 deletion in mice triggers diastolic dysfunction, 1 extracellular matrix response and impaired resolution of inflammation

30 The mammalian circadian clock consists of multiple transcriptional regulators that 31 coordinate biological processes in a time-of-day-dependent manner. Cardiomyocyte- 32 specific deletion of the circadian clock component, Bmal1 (aryl hydrocarbon receptor 33 nuclear translocator-like protein 1), leads to age-dependent dilated cardiomyopathy and 34 decreased lifespan in mice. We investigated whether cardiomyocyte-specific Bmal1 35 knockout (CBK) mice display early alterations in cardiac diastolic function, extracellular 36 matrix (ECM) remodeling, and inflammation modulators by investigating CBK mice and 37 littermate controls at 8 and 28 weeks old ( i.e. , prior to overt systolic dysfunction). Left 38 ventricles of CBK mice exhibited (p<0.05): 1) progressive abnormal diastolic septal 39 annular wall motion and reduced pulmonary venous flow only at 28 weeks of age; 2) 40 progressive worsening of fibrosis in the interstitial and endocardial regions from 8 to 28 41 weeks of age; 3) increased (>1.5 fold) expression of collagen I and III, as well as the 42 matrix metalloproteinases MMP-9, MMP-13, MMP-14 at 28 weeks of age; 4) increased 43 transcript levels of neutrophil chemotaxis and leukocyte migration genes ( Ccl2, Ccl8, 44 Cxcl2, Cxcl1, Cxcr2, Il1 β ) with no change in Il-10 and Il-13 genes expression; and 5) 45 decreased levels of 5-LOX, HO-1 and COX-2, enzymes indicating impaired resolution of 46 inflammation. In conclusion, genetic disruption of the cardiomyocyte circadian clock 47 results in diastolic dysfunction, adverse ECM remodeling, and pro-inflammatory gene 48 expression profiles in the mouse heart indicating signs of early cardiac aging in CBK 49 mice.


Introduction
transcription factors, CLOCK and BMAL1, has been investigated (42). These studies 82 reveal important roles for these clock components in the heart at the levels of 83 contractility, electrophysiology, metabolism, signaling, translation, and transcription (7). 84 Of these models, genetic ablation of BMAL1 in the heart elicits the most striking 85 phenotype from 28 week onwards; age-onset development of dilated cardiomyopathy 86 associated with reduced life span. The mean survival for cardiomyocyte specific Bmal1 87 deletion (CBK) mice was 33 weeks, while no deaths were observed in littermate 88 controls (i.e., Bmal1 flox/flox ) or wild-type (C57BL/6J) within one year of the study (42). 89 The mechanism(s) by which genetic ablation of Bmal1 in the heart results in 90 cardiomyopathy and an accelerated aging phenotype is (are) currently unknown. We 91 determined that Bmal1 deletion initiates an extracellular matrix (ECM) response which 92 proceeds to an inflammatory response by 28 weeks of age. Compared to littermate 93 controls, germline Bmal1 deletion results in the development of chronic inflammation (6). 94 Here, we report that ECM genes are increased in CBK mice at the age of 28 weeks. As 95 expected, with increased ECM deposition, the transforming growth factor (TGF-β) 96 signaling cascade, including SMAD2 (Mothers against decapentaplegic homolog 2), 97 was activated in CBK hearts by 28 weeks of age. Thus, Bmal1 deletion in mice initiated 98 dilative hypertrophy, diastolic dysfunction, and ECM responses and an impaired 99 resolution of inflammation axis with marked signs of accelerated early cardiac aging by Mice were anesthetized with 2% isoflurane in an oxygen mix and injected with 4 IU/g 133 heparin. After 5 minutes, the carotid artery was cut and plasma from the blood was 134 collected. The left ventricle (LV) was injected with 2 ml cardioplegic solution to arrest 135 the heart in a diastolic state, the whole heart and lungs were then removed. The right 136 ventricle and LV were separated. Lungs were removed and all masses were recorded. 137 The tibia was removed and placed in 1.5 M potassium hydroxide to digest for 24 hrs 138 and the length was measured with a vernier caliper. The LV was cut into 3 sections, 139 with the middle being stored in 10% formalin for 24 hours, then preserved in 70% 140 ethanol for histological analysis and the remaining LV sections are snap frozen and 141 stored at -80 C o for further cellular and molecular analysis.

197
Densitometry was performed using Image J software. (unpaired) was applied. p<0.05 was considered as statistically significant.

206
To determine if CBK mice develop age-dependent changes in LV mass, we measured 207 the weights of the right and left ventricles at 8 and 28 weeks. At 28 weeks of age, CBK 208 mice had higher RV mass, RV/body weight and larger LV mass/ body weight and LV 209 mass/tibia ratios than littermate control 28 week old controls (Table 1); in contrast, no 210 genotype-dependent effects were observed at 8 weeks of age. LV cardiac hypertrophy 211 was next assessed by measuring myocyte area cross-sections using WGA staining.

212
Myocyte area was not different between control and CBK mice at 8 weeks of age 213 (Figure 1a and b). However, at 28 weeks of age, CBK mice showed a 3 fold increase in 214 the surface area of myocyte, indicative of cardiomyocyte hypertrophy (Figure 1a and b).

215
CBK mice develop age-dependent diastolic dysfunction 216 mice (42). To determine whether development of diastolic dysfunction occurred at a 219 younger age in CBK mice, we measured diastolic function in CBK and control mice 220 using doppler echocardiography at 8 and 28 weeks. At 8 weeks of age, no significant 221 differences were observed for markers of diastolic function between control and CBK 222 mice (Table 2). However, at 28 weeks of age, CBK mice showed changes in several 223 key functional parameters, including increased mitral valve E/A ratio, abnormal diastolic 224 septal annular wall motion and reduced pulmonary venous flow (Table 2; Figure 1c).

225
Mitral valve E/A ratio was ~1.5 in control mice at both 8 weeks and 28 weeks, in 226 comparison, the E/A ratio was progressively increased >2 from 8 weeks to 28 weeks in  CBK mice displayed higher collagen content, accelerated fibrosis and ECM gene 285 response in mRNA levels, which was initiated as reparative fibrosis by 8 weeks of age, and progressed to reactive pathological fibrosis by 28 weeks of age. In order to confirm 287 these changes at the protein level at 28 weeks, we performed immunoblotting. Protein 288 analysis revealed an increase in the expression levels of collagen I & III in CBK hearts 289 (Figure 4a-e). Similarly, matrix metalloproteinase-9 (MMP-9) with TIMP-1 were 290 increased in CBK hearts at 28 weeks of age.

CBK hearts showed impaired resolution of inflammation with aging 292
Given that cardiac dysfunction is often associated with delicate imbalances in pro-293 inflammatory and resolution of inflammation mechanisms, an array of inflammatory 294 transcripts were determined in control and CBK hearts at 8 and 28 weeks of age. indicating that altered circadian clock from Bmal1 deletion leads to an impaired 321 inflammation resolution axis compared with control mice.

323
Central and peripheral circadian clock influence multiple aspects of cellular physiology, 324 and as a consequence, disruption of clock function invariably results in cardiovascular 325 pathology (11,32). In this study, we defined the role of Bmal1 in age-related LV 326 remodeling and diastolic dysfunction, through characterization of control and CBK mice 327 at 8 and 28 weeks of age. Importantly, the present study was aimed towards addressing 328 the role of Bmal1 in age onset cardiac remodeling rather than time of day events. Our 329 results highlighted that cardiac-specific deletion of Bmal1 in mice; 1) initiated dilative  In CBK mice, increased LV mass to body weight ratio is indication of cardiac 351 hypertrophy which occurs due to continuous pressure load that may transit to heart 352 failure due to diastolic dysfunction, systolic dysfunction, or combination of both. CBK cardiomyocyte size indicating age-associated hypertrophy at 28 weeks in CBK mice (scale bars: 582 10 μm). b) Quantitation of cardiomyocyte area/section/mouse indicated hypertrophic response 583 at 28 weeks of age in CBK mice. *p<0.05 vs age-matched control (CON); n=5 mice/group. c) 584 Representative echocardiographic images with abnormal diastolic septal annular wall motion 585 indicating diastolic dysfunction in CBK mice at 28 weeks compared with age-matched control. 586