Cardiomyocyte Cyclin-dependent kinase 9 directly binds to and phosphorylates NF-κB p65 subunit to drive cardiac inflammation and remodeling

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
Hypertensive heart failure highlights an urgent need for effective therapeutic strategies. Protein kinases regulate multiple pathways in cardiac pathophysiology and may provide promising therapeutic targets. Here, we identified a Cyclin-dependent kinase, CDK9, promoting inflammation and cardiac remodeling in terminally differentiated cardiomyocytes. Firstly, kinase enrichment analysis and experimental evidence revealed CDK9 phosphorylation at Thr-186 in both human and mouse hypertrophic heart tissues. CDK9 loss of function via T186A mutation in cardiomyocytes attenuated Ang II-induced heart remodeling and NF-κB-mediated inflammation, whereas CDK9 overactivation by T186E mutation induces. This regulatory function of CDK9 in cardiac remodeling is cell cycle-independent. Further studies demonstrate that the kinase domain of CDK9 directly binds to NF-κB P65 protein, which leads to the CDK9/P65 complex nuclear translocation, P65 phosphorylation, and transcription of inflammatory and hypertrophic genes in cardiomyocytes. This process requires CDK9 Thr-186 phosphorylation and Cyclin T1 presence, but is independent on IKKβ and CDK9-RNAPII pathways. Pharmacological inhibition of CDK9 phosphorylation significantly attenuated Ang II-induced cardiac inflammation, remodeling, and dysfunction in mice. Collectively, Ang II-activated CDK9 directly binds to and phosphorylates P65 to drive cardiac inflammation and remodeling. This study identifies CDK9 as a potential target in heart failure therapeutics.

Introduction
Prolonged and poorly controlled hypertension progressively develops into myocardial systolic hypokinesis, ventricular remodeling and heart failure, which is the leading cause of cardiovascular diseases (CVD) and death worldwide1,2. Abnormal elevation of angiotensin II (Ang II) leads to hypertension, initiates cardiac inflammation3 and eventually develop into ventricular dilatation4 and heart failure with reduced ejection fraction (HFrEF)5. Although the anti-hypertrophic drugs were applied to control the blood pressure, hypertensive CVD incidence remains high, indicating Ang II-induced other pathological pathways critically contribute to hypertensive complications. Therefore, finding the regulatory molecules of Ang II-induced cardiomyocyte injuries may provide targets for the treatment of hypertensive cardiac hypertrophy.

Serine/threonine kinases modulate a wide range of cellular events under normal conditions and in pathological states6,7. Small-molecule inhibitors of serine/threonine kinases have proven efficacy for the treatment of oncological disorders. Identifying serine/threonine kinases involved in pathological cardiac remodeling and exploring their function and mechanism may provide insights and therapeutic targets for hypertensive heart failure. Interestingly, as pro-phosphorylating enzymes, serine/threonine kinases may predominantly undergo alterations in the kinase activity while maintaining unchanged expression levels in diseases. In the present study, we profiled kinase activation in heart tissues of mouse and human models using Kinase Enrichment Analysis (KEA) based on the published RNA-Seq databases and identified increased phosphorylation level of Cyclin-dependent kinase 9 (CDK9) in hypertrophic hearts and cardiomyocytes.

CDK9 is an intracellular kinase regulating target gene transcription. Unlike cell cycle CDKs, the expression of CDK9 and its cyclin partner, as well as its kinase activity, do not change in a cell cycle-dependent manner8,9. CDK9 indirectly affects the expression of cell cycle-associated proteins by regulating transcription elongation10. Canonically, CDK9 binds to Cyclin T1 to form the complex of CDK9/Cyclin T1, known as positive transcription elongation factor b (P-TEFb), which triggers the RNA polymerase II (RNAPII) from pause to launch to drive the gene transcription11. CDK9 overactivation is closely implicated in the development of multiple chronic diseases12. In cancer, CDK9 modulates the transcription of multiple proto-oncogenes and inhibition of CDK9 is identified as the promising therapeutic strategy in cancer treatment13. Besides, CDK9 also triggers the transcription of fibrotic genes and regulates the nonalcoholic steatohepatitis progression through mediating the phosphorylation of METTL314, and controls LPS-induced macrophage inflammatory gene expression by JAK2-STAT3 signaling pathway15. Blocking CLP-1 binding to P-TEFb and inhibiting CDK9 kinase activity in fetal mouse heart resulted in a state of de-inhibited kinase activity of P-TEFb, which stimulates a hyper-responsive state of cardiomyocytes to hypertrophic signals16. However, the role and regulating mechanism of CDK9 in terminally differentiated cardiomyocytes remain unclear, and whether CDK9 mediates the hypertensive cardiac remodeling is largely unknown.

Here, we investigated the function of CDK9 phosphorylation in cultured cardiomyocytes and murine models of Ang II-induced cardiac remodeling. We demonstrated that Ang II activated the phosphorylation of CDK9 at Thr-186, which result in recruitment and interaction of NF-κB P65 subunit to activate NF-κB and induce the inflammatory and hypertrophic genes transcription in cardiomyocytes. Formation of the CDK9-P65 complex represents a mechanism of transcriptional regulation in pathological cardiac remodeling. We also showed that pharmacological inhibition of CDK9 significantly protects hearts in Ang II-infused mice.

Results
CDK9 phosphorylation, but not expression level, is increased in hypertrophic heart tissues
To find the changed kinase signaling pathway contributing to heart failure in an unbiased manner, we analyzed four RNA-Seq databases from mouse models with pathological cardiac remodeling and collected the up-regulated gene sets for Kinase Enrichment Analysis (KEA). As shown in Figure 1A and Supplementary Data 1, we found 46 kinases were activated in these four models. Furthermore, by cross-analysis with the KEA on the RNA-Seq database of human hypertrophic heart samples, we finally identified 5 activated candidate kinase signals involved in cardiac remodeling (Figure 1A, Supplementary Figure 1A-D). Next, we established a cardiac remodeling in mice through Ang II infusion for 4 weeks (Figure 1B)17, and harvested heart tissues to confirm the activation of these 5 kinases. We selected the antibodies of these 5 kinases specifically targeting their phosphorylated activating forms, respectively. For example, the antibody for p-CDK9Thr-186 was used because phosphorylation at this site is a key determinant of CDK9 kinase activity18. As shown in Figure 1C, we found that CDK9 phosphorylation was the most significant among these five kinases, indicating the pathogenic role of CDK9 phosphorylation in cardiac remodeling. Besides, we observed that Ang II challenge significantly weakened the association of CDK9 with the inhibitory 7SK snRNA complex (components LARP7 and HEXIM1), while concurrently strengthened its interaction with the activating partner Cyclin T1, validating the Ang II-induced CDK9 activation in cardiomyocytes (Supplementary Figure 1E-I).

In addition, the basal protein and gene expression levels of CDK9 were not altered in the mice with heart failure (Figure 1C, Supplementary Figure 1J). Similarly, we found the level of CDK9 phosphorylation in human hypertrophic heart was also significantly increased compared to the healthy donor, while the basal CDK9 protein level showed no change (Figure 1D). Parallelly, we examined p-CDK9/CDK9 level and distribution in human hypertrophic heart tissues by using the immunofluorescence staining, confirming CDK9 activation in hypertrophic human heart. The immunofluorescence double-staining also showed that the cell type expressing CDK9 most is cTNT-positive cardiomyocyte, rather than the Vimentin-positive fibroblasts (Figure 1E-F). Similar results were observed by the immunofluorescence double-staining using the heart tissues from mouse model (Supplementary Figure 2A-B), indicating CDK9 activation occurs mainly in cardiomyocytes. Further, we exposed the neonatal rat ventricular myocytes (NRVMs), a well-established in vitro cardiomyocyte model19, to 1 μM Ang II and observed that the phosphorylation of CDK9 increased time-dependently and peaked at 6 h, while Ang II stimulation did not affect the total CDK9 level in cultured cardiomyocytes (Supplementary Figure 2C). In addition, CDK9 dissociates from 7SK RNA and transitions into an activated state in Ang II-treated NRVMs (Supplementary Figure 2D-H). We also explored how Ang II induces CDK9 activation. We found that AT1R knockdown attenuated the CDK9 phosphorylation induced by Ang II in NRVMs, indicating that CDK9 is one of the downstream signals of Ang II-AT1R axis (Supplementary Figure 3A-B). These findings indicate that CDK9 phosphorylation was induced by Ang II in hypertrophic heart tissues and cardiomyocytes.

CDK9 phosphorylation at Thr-186 mediates Ang II-induced remodeling in cardiomyocytes
The specific CDK9 phosphorylation inhibitor20, NVP2, and CDK9-siRNA (siCDK9) were utilized to study the effects of CDK9 phosphorylation in Ang II-induced cardiomyocyte injury. Firstly, the NVP2 shown no influence on the cell cycle in NRVMs (Supplementary Figure 4A), and the dosage of NVP2 at 1 μM was chosen for the further study. NVP2 treatment indeed blocked the Ang II-induced CDK9 phosphorylation in NRVMs (Supplementary Figure 4B-C). Then, NVP2 alleviated Ang II-induced overexpression of genes/proteins related to hypertrophy and fibrosis (Figure 2A-B and Supplementary Figure 4D). In addition, knockdown of CDK9 in NRVMs also attenuated Ang II-increased these genes/proteins. (Figure 2C-D, Supplementary Figure 5A-B). Meanwhile, either inhibited or down-regulated CDK9 reduced the enlarged cardiomyocytes size induced by Ang II (Figure 2E and Supplementary Figure 5C). Collectively, CDK9 mediates the pathological remodeling in Ang II-challenged NRVMs.

The phosphorylation sites in CDK9 contains Ser-90, Ser-175, Thr-186, Ser-347, Thr-262, and Thr-363. Considering the Ser-9021 and Ser-17522 are related to facilitating CDK9 T-loop interactions with Tat and HIV transcription, we focused more on the Thr-186 in CDK911,23. To explore the effects of CDK9 Thr-186 phosphorylation in NRVMs specifically, we made a point mutation for Thr-186. Two separate mutation plasmids, HA-tagged CDK9 (Wild type, CDK9WT) and HA-tagged CDK9 with Thr-186-Ala (inactivation, CDK9T186A) were conducted (Supplementary Figure 6A). We next transfected the CDK9WT and CDK9T186A into the CDK9-deficient NRVMs (transfected with siCDK9 for 24 h) and then exposed to Ang II for 24 h. As shown, inactivation of 186-site abolished Ang II-induced phenotypes of hypertrophy and fibrosis in cardiomyocytes (Figure 2F-H and Supplementary Figure 6B-C), indicating that Thr-186 phosphorylation is specifically responsible to Ang II-induced cardiomyocyte injury. On the other hand, a self-activating mutation plasmid, HA-tagged CDK9 with Thr-186-Glu (self-activation, CDK9T186E) was used to simulate the CDK9 phosphorylating activation in NRVMs (Supplementary Figure 7A). As shown in Supplementary Figure 7B-F, compared to T186A, the T186E mutant reduced CDK9 interaction with HEXIM1 and enhanced its binding to Cyclin T1. Consistently, self-activation of CDK9 caused hypertrophy and fibrosis phenotypes in NRVMs without Ang II stimulation (Figure 2I-J and Supplementary Figure 7G-J), and over-activation of CDK9 in Thr-186 cancelled the pro-hypertrophic effect of Ang II (Figure 2I-J). In conclusion, these data demonstrated the Thr-186 phosphorylation of CDK9 mediates Ang II-induced cardiomyocyte remodeling.

NF-κB mediates cardiomyocyte remodeling induced by Ang II-CDK9 signal
To explore the downstream mechanism of Ang II-CDK9 signal, a transcriptome RNA-seq was performed in the NC and siCDK9 NRVMs with Ang II challenging. As shown in Figure 3A-B and Supplementary Figure 8A-B, 520 DEGs were down-regulated in NC-Ang II group vs siCDK9-Ang II group, and the enrichment pathway analysis showed that these down-regulated genes were involved in NF-κB signaling. Since NF-κB has been demonstrated as a critical transcriptional factor regulating genes related to inflammation and remodeling in cardiomyocytes24,25, we considered NF-κB signaling as the potential mechanism participating in CDK9-mediated cardiomyocyte injury. Next, several assessments were performed to validate this hypothesis. CDK9 deficiency and inhibition relieved the NF-κB P65 phosphorylation and nuclear translocation induced by Ang II in NRVMs (Figure 3C-D and Supplementary Figure 8C-F). Further, the reported gene fluorescence intensity showed that silencing or inhibiting CDK9 attenuated Ang II-stimulated the transcriptional activity of NF-κB P65 (Figure 3E). CHIP-qPCR also showed the decreased level of P65 binding with the Il6 promotor when CDK9 was knocked down (Figure 3F), parallel with the inhibition of these Ang II-activated inflammatory gene transcription (Supplementary Figure 8G).

We then ascertained whether Thr-186 phosphorylation of CDK9 mediated NF-κB activation. Similarly, inactivating mutation of Thr-186-Ala attenuated Ang II-induced P65 nuclear translocation and inflammation response in NRVMs, while the self-activation of CDK9 by CDK9T186E mutation significantly induced NF-κB activation and inflammatory gene transcription (Figure 3G-J and Supplementary Figure 8H-K). Moreover, P65 deficiency by siRNA blocked the inflammatory and remodeling-related gene expression induced by CDK9T186E self-activation in NRVMs, indicating that NF-κB is a downstream effector mediating CDK9’s inflammatory response. (Figure 3K-L and Supplementary Figure 9A-C). These data validate that CDK9 Thr-186 phosphorylation mediates Ang II-induced NF-κB activation and subsequent inflammatory response and remodeling in NRVMs (Figure 3M).

Loss of Thr-186 phosphorylation of CDK9 attenuated the Ang II-induced cardiac remodeling and inflammation in mice
To investigate the function of cardiomyocyte CDK9 Thr-186 phosphorylation in heart remodeling, we constructed cardiomyocyte-specific CDK9 loss of function by injecting AAV9. Briefly, CDK9 was first knocked down in mouse cardiomyocytes by tail vein injection of AAV9-cTNT-GFP-CDK9shRNA, and then reconstructed the wild-type CDK9 or T186A mutant CDK9 by tail vein injection of AAV9-cTNT-HA-CDK9WTOE or AAV9-cTNT-HA-CDK9T186AOE, respectively (Supplementary Figure 10A-F). Following confirmation of successful AAV9 expression in the heart, these mice were received Ang II subcutaneous injection for 4 weeks to induce cardiac remodeling (Figure 4A).

As shown in Figure 4B and Supplementary Figure 11A, loss phosphorylation of CDK9Thr-186 did not affect the systolic blood pressure elevation, body weight change and heart rate in Ang II-infused mice. Echocardiography was performed to assess the cardiac function, and CDK9 Thr-186 inactivation attenuated Ang II-induced heart dysfunction and preserved the normal size of heart (Figure 4C and Table 1). Besides, CDK9 function loss decreased the Ang II-increased cardiomyocyte area, maintained tight cell structure and alleviated the heart fibrosis in mice (Figure 4D and Supplementary Figure 11B-C). Parallelly, the protein and mRNA levels of heart remodeling markers were also significantly reduced in mutant CDK9 group, compared to the WT CDK9 group followed Ang II infusion (Figure 4E-I and Supplementary Figure 11D-G). Cardiac inflammation was also inhibited, as evidenced by that CDK9 Thr-186 mutation blocked the inflammatory gene expression and NF-κB P65 pathway activation in Ang II-challenged mouse heart (Figure 4J-O and Supplementary Figure 11H-K). Collectively, these results confirmed that loss of Thr-186 phosphorylation in CDK9 in cardiomyocytes attenuated the Ang II-induced cardiac remodeling and inflammation.

Self-activated Thr-186 phosphorylation of CDK9 in cardiomyocytes induced cardiac remodeling and inflammation in mice
Furthermore, we assessed the effect of activated Thr-186 phosphorylation of CDK9 in cardiomyocytes on cardiac remodeling. Similarly, by tail vein injection of AAV9-cTNT-HA-CDK9T186EOE or AAV9-cTNT-HA-CDK9WTOE into mice with CDK9 pre-knockdown in cardiomyocytes, we obtained cardiomyocyte-specific CDK9 autophosphorylation-activated mice, as well as the control mice (Figure 5A).

After 8-week feeding, these mice were evaluated for heart function and then euthanized. Autophosphorylation of CDK9 at T186 in cardiomyocytes shown no influence in systolic blood pressure, body weight alteration and heart rate of mice (Figure 5B, Supplementary Figure 12A). However, persistent CDK9 activation in cardiomyocytes induced cardiac hypertrophy (Figure 5C) and dysfunction (Figure 5C, Table 2). Microscopically, the area of cardiomyocytes was increased, and cardiomyocytes were loosely arranged and the fibrosis was occurred in hearts of CDK9T186EOE mice (Figure 4D-F). CDK9 self-activation in cardiomyocytes stimulated the overexpression of genes related to cardiac hypertrophy and fibrosis in mouse heart (Figure 5G-K and Supplementary Figure 12B-E), and also promoted the levels of inflammatory factors, accompanied with the hyperactivation of NF-κB P65 pathway (Figure 5L-R and Supplementary Figure 12F). Combined with the findings above, we conclusively demonstrated that CDK9 phosphorylation at T186 in cardiomyocytes positively regulates the process of cardiac remodeling through activating NF-κB signaling pathway.

CDK9 directly binds to P65 and Ang II challenge increases CDK9-P65 interaction in cardiomyocytes
To identify CDK9 substrate proteins, we conducted Co-IP coupled with mass spectrometry in both 293T cells and NRVMs, each transfected with the HA-CDK9 plasmid. Integrated analysis of the proteomic data with the top 10 signaling pathways previously annotated in Figure 3B revealed RELA (P65) as a candidate CDK9-binding protein (Figures 6A and Supplementary Figure 13A). We also performed a phosphoproteomics analysis on cardiac tissues from the Ang II and Ang II+NVP2 groups. The phosphorylation levels of both CDK9 and P65 were significantly suppressed by NVP2 treatment (Figure 6B). Reactome pathway enrichment analysis of NVP2-downregulated phosphoproteins revealed that NVP2 treatment markedly affected immune-response-related pathways (Supplementary Figure 13B). Furthermore, protein-protein interaction (PPI) analysis of the phosphoproteomics data indicated an interaction between CDK9 and P65 (Supplementary Figure 13C). These results suggest that CDK9 may directly interact with P65 to regulate its phosphorylation in cardiomyocytes.

Next, using Bio-Layer Interferometry (BLI) assay, a binding affinity was measured for rhCDK9 (recombinant human CDK9) and rhP65 (recombinant human P65) interaction and KD (the equilibrium dissociation constant) was determined to be 1.433 μmol/L (Figure 6C), validating that CDK9 directly binds to P65. Subsequently, we examined the endogenous CDK9-P65 interaction in NRVMs by co-immunoprecipitation assay, while Ang II challenge for 3-9 hours significantly increased the CDK9-P65 interaction (Figure 6D-E). Similar results were also observed in the 293T cells co-transfected with the plasmids of Flag-P65, HA-CDK9 and His-Cyclin T1, and followed with the Ang II stimulation (Figure 6F, Supplementary Figure 13D-E). Phosphorylated IKKβ (p-IKKβ) is known to directly phosphorylate IκBα, leading to its proteasomal degradation and subsequent liberation of free NF-κB p65 subunits. We examined CDK9's effect on IKKβ phosphorylation. As expected, neither CDK9 knockout nor pharmacological inhibition attenuated Ang II-induced IKKβ phosphorylation (Supplementary Figures S13F-G). Besides, silencing IKKβ did not alter Ang II-mediated CDK9 phosphorylation and CDK9-P65 complex formation (Supplementary Figures S13H-K). These results validate that CDK9 directly binds to and phosphorylates p65 through an IKKβ-independent mechanism.

We investigated the time course and subcellular redistribution of the CDK9-p65 complex upon Ang II stimulation in NRVMs using proximity ligation assay (PLA). The PLA signals, indicating endogenous CDK9-p65 interaction, were increasingly detected in the nucleus within 6-hour Ang II treatment, demonstrating that Ang II induces the formation and nuclear translocation of the CDK9-p65 complex (Figure 6G-H). Co-immunoprecipitation assay in NRVMs also confirmed the location shift of CDK9-P65 complex following the Ang II stimulation (Figure 6I, Supplementary Figure 13L-M). We then investigated the domains in CDK9 structure responsible for CDK9-P65 interaction. The structural sequence of CDK9 protein is shown in Figure 6J26, and four mutant CDK9 plasmids with the respective region deletion were designed and successfully expressed in 293T cells. Through co-transfection with Flag-P65 and HA-tagged mutant CDK9 plasmids, we found that lacking of the kinase domain (19aa-165aa and 192aa-315aa) decreased the interaction of CDK9 and P65, indicating that the kinase domain of CDK9 contributes to the CDK9 binding to P65 (Figure 6K).

Ang II-increased CDK9-P65 interaction depends on CDK9T186 phosphorylation and Cyclin T1
We then explored how Ang II increased CDK9-P65 interaction. The T-loop domain in CDK9 has been reported as the key region recognizing and binding to CyclinT1, which promotes CDK9 phosphorylation and activation11. We also assessed whether the CDK9-P65 interaction requires CyclinT1. As shown, knockdown of CyclinT1 reduced the CDK9-P65 complex formation, and inhibited the CDK9 and P65 phosphorylation (Figure 7A-B, Supplementary Figure 14A-C). We then examined if CDK9 phosphorylation increases CDK9-P65 interaction. Interestingly, both CDK9 inhibition and Thr-186 inactivation abolished Ang II-induced CDK9 binding to P65, while Thr-186 self-activation increased CDK9-P65 interaction in NRVMs (Figure 7C-E, Supplementary Figure 14D-F). These data indicate that Ang II-increased CDK9-P65 interaction depends on CDK9T186 phosphorylation and presence of Cyclin T1.

CDK9/Cyclin T1 has been reported to phosphorylate RNAPII and drive the transcription of downstream genes such as Myc27. We then tried to examine if P65 is involved in CDK9-RNAPII signaling. As seen in Figure 7F and Supplementary Figure 15A-D, P65 knockdown showed no alteration on Cyclin T1-CDK9-RNAPII complex formation and RNAPII phosphorylation in NRVMs. Also, P65 silence did not affect RNAPII binding to Myc promotor (Supplementary Figure 15E). In another word, the CDK9-P65 axis triggering inflammation response in cardiomyocytes was independent on the CDK9-RNAPII pathway. An illustrative diagram including this molecular event flow and the predictive CDK9-P65 molecular docking is shown in Figure 7G. These results revealed a potential event flow, in which CyclinT1-CDK9 interaction leads to CDK9 phosphorylation, then p-CDK9-P65 interaction and nuclear translocation, and finally P65 activation.

Administration of CDK9 kinase inhibitor NVP2 protects hearts against inflammation and remodeling in mice.
For the translational perspective, we tested whether pharmacological inhibition of CDK9 kinase using NVP2 could preserve heart from Ang II-induced cardiac pathology in vivo. Ang II-challenged mice were administrated with NVP2 (5 mg/kg/day, intraperitoneally) or vehicle for the final 2 weeks. The dosage of NVP2 used was decided according to the previous study20,28. No significant body-weight loss and toxicity in NVP2-treated mice were observed during the 4-week study (Supplementary Figure 16A). Also, NVP2 did not affect systolic blood pressure and heart rate in Ang II-infused mice (Supplementary Figure 16B). Using the echocardiography, we observed that NVP2 attenuated several hallmark features of cardiac dysfunction in Ang II-infused mice (Figure 8A, Supplementary Table 1). NVP2 treatment also protected mice from Ang II-induced heart enlargement, cardiomyocyte hypertrophy, and ventricular fibrosis (Figure 8A-B, Supplementary Figure 16C-E). Consistently, several markers of cardiac remodeling in protein and mRNA levels were also reversed by NVP2 treatment compared to the Ang II-infused group (Figure 8C-D, Supplementary Figure 16F-G). These data demonstrate that NVP2 attenuates Ang II-induced cardiac remodeling and dysfunction in mice.

We then examined the CDK9-P65 axis and inflammatory response in mouse hearts. The anti-inflammatory effect of NVP2 in heart was confirmed by the decreased TNFα expression in heart tissues and the reduced transcription of the pro-inflammatory genes (Figure 8E, Supplementary Figure 17A-B). This anti-inflammatory effect was associated with the inhibition of CDK9 phosphorylation in NVP2-treated mouse heart (Figure 8F, Supplementary Figure 17C). Inhibition of CDK9 also attenuated Ang II-induced P65 phosphorylation (Figure 8G, Supplementary Figure 17D-E) and formation of CDK9-P65 complex in heart tissues (Figure 8H-I). As expected, NVP2 showed no inhibition on Ang II-induced IKKβ phosphorylation in mouse hearts (Supplementary Figure 17F). These results demonstrate that pharmacological inhibition of CDK9 blocked the CDK9-P65 axis and attenuated inflammation in hearts.

Methods
General Reagents
Ang II peptide (#A107852) was purchased from Aladdin Chemicals (Shanghai, China). CDK9 inhibitor (NVP2) was purchased from Selleck (#S8981, Shanghai, China). Recombinant human CDK9 (rhCDK9) was purchased from Solarbio (#P02552; Beijing, China). Recombinant human P65 (rhP65) was obtain form the CBRC of Wenzhou Medical University with a purity>95%. Antibodies against CDK9 (#2316), Phospho-CDK9 (Thr-186) (#2549), Cyclin T1(#81464), RNAPII (#2629), Phospho-RNAPII (at Ser2/5 in C-terminal domain) (#4735), NF-κB p65 (#8242T), Phospho-NF-κB p65 (Ser536) (#3033), IκB kinase-beta (IKKβ) (#8943), Phospho-IKKα/β (Ser176/180) (#2697), Phospho-c-Src (Tyr416) (#6943), Phospho-PKA C (Thr197) (#5661), HEXIM1(#12604T), PKC-alpha (#2056), Phospho-PKC alpha (Thr638) (#9375) and Lamin B1 (#13435) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Cardiac Troponin T (cTNT, #ab8295) and LARP7 (ab134757) was obtained from Abcam (Shanghai, China). Antibodies against angiotensin receptor AGTR1 (#sc-515884) and Vimentin (#sc-6260) were obtained from Santa Cruz Biotech (Texas, USA). Antibodies against cardiac myosin heavy chain-7 (MYH7, #22280-1-AP), collagen type I (COL1a1, #67288-1-Ig), HA-tag (#51064-2-AP), AGTR1 (#25343-1-AP), transforming growth factor-β1 (TGF-β1, #21898-1-AP), TNF-α (#60291-1-Ig), Flag-tag (#20543-1-AP), Caspase3/p17/19(#19677-1-AP) and GAPDH (60004-1-Ig) were purchased from Proteintech (Shanghai, China). Antibodies against cGKI (phospho-Thr515) (#orb182769) and Hck (phospho-Tyr411) (#orb184018) were purchased from Biorbyt (Wuhan, China). Alexa Fluor 594-conjugated wheat-germ agglutinin (WGA, #W11262) was purchased from Thermo Fisher (Waltham, MA, USA). Rat IgG2a Isotype (#65209-1-Ig) was purchased from Proteintech (Shanghai, China). Rhodamine phalloidin (#CA1610) were purchased from Solarbio (Beijing China). Secondary anti-rabbit IgG HRP (#7074), anti-mouse IgG HRP (#7076) and anti-rat IgG HRP (#7077) were purchased from Cell Signaling Technology. Goat anti-mouse IgG alexa fluor 488 (#A-11001) and donkey anti-rabbit IgG alexa fluor 568 (#A10042) were purchased from Thermo Fisher.

Human subject collection
In this study, 2 male patients with heart failure in the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine for heart surgical treatment were enrolled. All diagnosis were made according to 2021 ESC Guidelines for heart failure48. The 2 control hearts tissues were obtained during the mitral valve replacement surgery with the health cardiac function. Written informed was obtained from the patients or family members. This study and all protocols used were approved by the Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (Ethics number: 2023-433-01) and conformed to the principles outlined in the Declaration of Helsinki.

Cell culture studies
Human embryonic kidney cell line HEK-293T was obtained from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). HEK-293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco/BRL life Technologies, Eggenstein, Germany) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 U/mL streptomycin.

The isolation and culture of neonatal rat ventricular myocytes (NRVMs) were performed as described previously49. To enrich cardiomyocytes, we utilized the principle of differential adhesion, whereby non-cardiac cells (primarily fibroblasts) adhere more rapidly to culture dishes than cardiomyocytes. Briefly, heart tissues of neonatal Wistar rats were dissociated with trypsin and cells were plated for 1 hour into tissue culture dishes in DMEM with 10% FBS to reduce the number of nonmyocytes. During this short incubation, the majority of fibroblasts attached to the dish, while cardiomyocytes remained largely in suspension. NRVMs that did not attach to the plates were aspirated and plated into 6-well culture plates. Primary cardiomyocytes were cultured in the same growth medium in DMEM (Gibco/BRL life Technologies, Eggenstein, Germany) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 U/mL streptomycin.

Gene knockdown and overexpression
Gene silencing was achieved by transfecting cells with siRNA (Gene Pharma). Custom siRNAs were synthesized for rat CDK9 (5'- GCCAGAAGGUGGCUCUGAATT-3' and 5'- UUCAGAGCCACCUUCUGGCTT-3'); rat P65 (5'- GAGUAGAGUCAGGAGGGCAGA-3' and 5'- UCUGCCCUCCUGACUCUACUC-3'); rat AT1R (5'- GGCAGGCACAGUUAC AUAUTT-3' and 5'-AUAUGUAACUGUGCCUGCCTT-3'); rat CyclinT1 (5'-GUCCAU UCUUAUGGUGAAUTT-3' and 5'- AUUCACCAUAAGAAUGGACTT-3'); rat IKKβ (5′‐ UUACUGAGCUGAGAGUAAAUCACCC‐3′, 5′‐GGGUGAUUUACUCUCAGCUCAGUA A‐3). Control cells were transfected with negative control siRNA. Transfection of NRVMs was carried out using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, 13778150). Overexpression plasmids Flag-P65 (HG12054-CF) and HA-CDK9 (HG10907-CY) were purchased from SinoBiological, and the mutant plasmids were designed and obtain from GeneChem. WT and mutant CDK9 overexpression, as well as P65 overexpression, in NRVMs and HEK-293T cells were achieved by transfecting cells with HA-tagged wildtype CDK9 (HA-CDK9wt), HA-tagged CDK9 with 19-165aa deficiency (HA- CDK9mut1), HA-tagged CDK9 with 166-191aa deficiency (HA-CDK9mut2), HA-tagged CDK9 with 192-315aa deficiency (HA-CDK9mut3), HA-tagged CDK9 with 345-372aa deficiency (HA-CDK9mut4). Transfection of primary macrophage was carried out using PEI Transfection Reagent according to the instruction (MCE, HY-K2014).

Flow cytometry
Cell cycle assays were performed on NRVMs according to the instructions of the Cell Cycle Staining Kit (CCS012, Multi-Science, China) and test by BD Fortessa (BD, USA).

Luciferase activity assay
NRVMs cultured in 6‐well plates were transfected with the pNFκB-TA-luc plasmids (D2207, Beyotime, Shanghai, China) and Renilla luciferase plasmids (control plasmids) (RG016, Beyotime, Shanghai, China) by PEI Transfection Reagent (HY-K2014, MCE, China) as indicated for 24 h. And then, NRVMs were transfected with control siRNA (NC), CDK9 siRNA (siCDK9) or pretreated with NVP2 (1 µM) for 1 h, respectively, and then exposed to 1 µM Ang II for 6 h. Luciferase activity was measured in the cell lysates by a dual luciferase reporter assay system.

Transcriptome sequencing
Total mRNA form NRVMs from siCDK9-Ang II vs NC-Ang II group were isolated using TRIZOL (Takara, Kyoto, Japan), and the transcriptome sequencing was conducted by BGI (WuHan, China). The differentially expressed genes (DEGs) were defined by fold change > 1.5 or < 0.67, and P < 0.05. Further analysis, including PCA analysis, drawing of Volcano plot and Venn diagram were conducted in Dr. Tom in BGI (WuHan, China). Enrichment of intersecting genes were then conducted in the database of “Elsevier Pathway Collection” in Enrichr software (https://maayanlab.cloud/Enrichr/)50 by using the raw values of RNA-seq data. The combined score was calculated as follows: c = log(p) · z, where c is the combined score, p is the p-value computed using the Fisher exact test, and z is the z-score computed by assessing the deviation from the expected rank. Raw data of transcriptome sequencing were deposited in the Sequence Read Archive (SRA) in BioProject ID: PRJNA1012513.

LC-MS/MS Analysis
CDK9 antibody was added to a sample of NRVMs respectively. Lysate for IP experiment, and IgG was used as a negative control. The lysate samples were collected from three independent biological replicates and pooled as one sample. Then, the LC-MS/MS analysis was carried out by AIMS Scientific Co., Ltd (Shanghai, China). Briefly, precipitated proteins were in-gel digested by adding 1% trypsin and incubating at 37 °C overnight into peptides. The mobile phase was prepared with Solution A (0.1% formic acid in MS water) and Solution B (100% acetonitrile). Peptides were separated with a linear gradient from 4% to 90% Buffer B over 98 min, and all at a constant flow rate of 600 nl/min on a Thermo Fisher Easy-nLC 1000 (USA). The peptides were ionized via a capillary source and analyzed using a Thermo Fisher Q Exactive HF-X (USA). Search the database using Proteome Discoverer 3.1 (Thermo, USA). Proteins identified with a minimum of two unique peptides were considered validly identified proteins. Potential substrate proteins that could interact with CDK9 were identified. Finally, we screened out the substrate proteins that could bind to CDK9 according to the score and the mass of detected proteins.

Immunofluorescence cell staining
For NF-κB (p65) translocation studies, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min and then blocked with 5% BSA in PBS for 30 min. Next, fixed cells were incubated with anti-NF-κB (p65) antibody (1:200) overnight at 4°C. Alexa fluor 488-conjugated secondary antibody (1:1000) was used for detection. Immunofluorescence was viewed and captured using Olympus fluorescence microscope. IgG staining was used as a primary antibody control.

To detect cellular hypertrophy, primary cardiomyocytes were exposed to Ang II, with or without CDK9 siRNA, NVP2 or other plasmids pre-treatment. Cells were then fixed and permeabilized as indicated above. Cells were stained with rhodamine phalloidin (Solarbio, CA1610) at a concentration of 100 nM for 30 min. Nuclei were stained with the DAPI (Sigma, D6578). Immunofluorescence images were obtained.

Computational docking and molecular simulation
HawkDock (http://cadd.zju.edu.cn/hawkdock/) methods is widely used to calculate the affinity of protein complex51. The crystal structures of human p65 (PDB:1NFI) and human CDK9 (PDB: 4EC8) were derived from RCSB PDB. The p65 was docked into the CDK9 to generate 100 binding poses. The binding free energy between each docking pose was scored by the MM/GBSA method in AmberTools package after a structure minimization52. Finally, based on the per-residue decomposition energy calculations, the key residues for protein-ligand interaction were identified.

Bio-layer interferometry binding assays
Binding kinetics measurements were performed using Bio-Layer Interferometry on FortéBio Octet Red 96 instrument. Purified kinase-active NFκB-P65 was biotinylated using Biotinylation Kit (G-MM-IGT, GENEMORE) following the manufacturer’s instructions. The biotin labeled-NFκB-P65 protein was loaded onto super streptavidin (SSA) sensors (18-5057, Octet) with 200 μL 25 mM HEPES, 150 mM NaCl, and 0.02% (v/v) Tween-20 as running buffer. After equilibrium, association and dissociation measurements were carried out using serial dilutions of rhCDK9 (62.5, 125, 250, 500, and 1000 nM) (P02552, Solarbio). The equilibrium constant (KD) values were assessed by GraphPad software 8.0.

In vivo randomization and blinding procedures
Sample sizes were defined by a priori power calculation with G-Power 3.1.9 software (http://www.gpower.hhu.de/), with a statistical power of 80% and α=0.05. We used a random number table to perform randomization, as described previously53. Briefly, all animal experiments were performed and analyzed in a blinded manner. Each mouse was assigned a temporary random number within the weight range. When mice were randomly divided in each group, they were given their permanent numerical designation in the cages. For each group, a cage was selected randomly from the pool of all cages. All data were collected and analyzed by two observers who were not aware of the group assignment or treatment of the mice.

Inclusion and exclusion criteria
Criteria for inclusions and exclusion were set before the study. Briefly, mice in the Ang II infusion group were included in the study if they underwent successful subcutaneous implantation of the Ang II mini-pump and had a significant increase in systolic blood pressure. Mice were excluded if systolic blood pressure did not rise after Ang II mini-pump implantation, or if the mini-pump was dislodged, or if the animal died prematurely, making it impossible to collect behavioral and histological data.

Mouse models of cardiac remodeling
All animal care and experimental procedures were approved by the Zhejiang Chinese Medical University Laboratory Animal Research Center and Welfare Committee (20201116-06). All animals received human care according to the National Institutes of Health (USA) guidelines and ARRIVE guidelines54. The male C57BL/6 mice weight 18-22 g were obtained from GemPharmatech (Nanjing, China). Mice were housed in a pathogen-free room under 22 ± 2°C, 50–60% humidity, 12:12 h light-dark cycle, and fed a standard rodent diet in Zhejiang Chinese Medical University Laboratory Animal Research Center. Mice were acclimatized to the laboratory for at least 2 weeks before initiating the studies.

Subcutaneous Ang II infusion by mini-pump was used to establish the mouse models of cardiac remodeling and dysfunction. Eight-week-old C57BL/6 mice were randomly divided into four groups: 1) controls mice with vehicle solution (Vehicle-Ctrl, n = 7); 2) Ang II-infused mice with vehicle solution (WT-Ang II; n = 7); 3) mice with NVP2 (NVP2-Ctrl, n = 7); and 4) Ang II-infused D1KO mice with NVP2 (NVP2-Ang II; n = 7). Ang II was administered using osmotic mini pump that delivered 1000 ng/kg/min (Alzet MODEL 1004, CA, USA) for 4 weeks, as described previously55. After 2 weeks of Ang II infusion, vehicle solution (5% DMSO in PBS, 0.1 mL) or NVP2 (5 mg/kg/d for 14 d, dissolved in 0.15 mL of the same vehicle solution) were started intraperitoneally until the end of Ang II infusion. At the end of treatment, mice were sacrificed under sodium pentobarbital for anesthesia and pain medication.

At the indicated time points, systolic blood pressure was measured by non-invasive tail-cuff Pressure Analysis System while mice were conscious (BP-98A; Softron, Tokyo, Japan). Measurements were performed during 1:00 pm to 5:00 pm with 5 days of previous training. When animals were sacrificed using sodium pentobarbital, the hearts were harvested and weighed to compare heart weight/body weight (HW/BW, mg/kg) ratios, and tibia lengths were measured to determine heart weight: tibia length ratios. Heart tissues were snap-frozen in liquid nitrogen for gene and protein expression analyses or fixed with 4% paraformaldehyde for histological analysis.

Adeno-Associated Virus Infection
To investigate the function of cardiomyocyte CDK9 Thr-186 phosphorylation in heart remodeling, adeno associated virus serotype 9 (AAV-9) targeted cardiomyocyte-CDK9 function were constructed by OBiO Technology (Shanghai, China), (1) AAV9-cTNT-GFP-CDK9shRNA: knockdown cardiomyocyte CDK9; (2) AAV9-cTNT-HA-CDK9WTOE: overexpress cardiomyocyte wildtype CDK9; (3) AAV9-cTNT-HA-CDK9T186AOE: overexpress cardiomyocyte CDK9 with Thr-186-Ala (inactivation, CDK9T186A); (4) AAV9-cTNT-HA-CDK9T186EOE: overexpress cardiomyocyte CDK9 with Thr-186-Glu (self-activation, CDK9T186E).

For the constructing mice with loss Thr-186 phosphorylation of CDK9 in cardiomyocytes. 21 wild type (WT) mice were divided into 3 groups (n = 7 per group) as follows: CDK9KO +CDK9WT-Ctrl, CDK9KO +CDK9WT-Ang II, CDK9KO +CDK9T186A-Ang II. The mice were injected with AAV9-cTNT-GFP-CDK9shRNA and AAV9-cTNT-HA-CDK9WTOE or AAV9-cTNT-HA-CDK9T186AOE, respectively via tail vein (2 × 1011 vg/mouse/month) for two months, followed by four weeks of Ang II stimulation according to previous research. Mice were sacrificed after assessment of cardiac function, and blood and heart tissues were collected for further analysis.

For the constructing mice with Self-activated Thr-186 phosphorylation of CDK9 in cardiomyocytes. 14 wild type (WT) mice were divided into 2 groups (n = 7 per group) as follows: CDK9KO +CDK9WT, CDK9KO +CDK9T186E. The mice were injected with AAV9-cTNT-GFP-CDK9shRNA and AAV9-cTNT-HA-CDK9WTOE or AAV9-cTNT-HA-CDK9T186EOE, respectively via tail vein (2 × 1011 vg/mouse/month) for two months, followed by four weeks rearing. Mice were sacrificed after assessment of cardiac function, and blood and heart tissues were collected for further analysis.

Cardiac functional tests
Systolic and diastolic cardiac function was determined non-invasively by transthoracic echocardiography in anesthetized mice, one day before the sacrifice as described previously56. Mice were anesthetized using isoflurane and echocardiography was performed by SONOS 5500 ultrasound (Philips Electronics, Amsterdam, Netherland) with a 15-MHz linear array ultrasound transducer.

Heart tissue staining
Hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Five μm thick sections were stained with hematoxylin and eosin (H&E) (Solarbio Life Sciences, Beijing, China), Picro Sirius Red, and Masson’s Trichrome (Solarbio Life Sciences, Beijing, China) for routine histology and assessment of cardiac fibrosis. The extent of fibrosis was assessed by calculating the percentage of fibrotic area relative to the total tissue area. For each tissue section (n=7), ten non-overlapping fields were randomly selected and examined under an optical microscope (Nikon, Japan). Quantification was subsequently performed using ImageJ software (version 1.38e).

For determination of cardiac myocyte cross-sectional areas, frozen heart sections were used. Briefly, portions of heart tissues were embedded in Optimal Cutting Temperature (OCT) media and sectioned at 5-μm thickness. Sections were stained with Alexa Fluor 594-conjugated wheat-germ agglutinin (1.0 mg/mL) and counterstained with DAPI. Images were captured using Nikon microscope (Nikon, Japan). Size of cells was measured.

For immunohistochemical staining, paraffin sections were deparaffinized and rehydration. Sections were treated with 3% H2O2 for 30 min to block endogenous peroxidase activity and then blocked with 1% BSA in PBS for 30 min. Slides were incubated overnight at 4°C with primary antibodies against TNF-α (1:50, 60291-1-Ig). Peroxidase-conjugated secondary antibodies were used for detection (1:500). Slides were counterstained hematoxylin for 5 min, dehydrated, and mounted. Images were viewed by a bright field microscope (Nikon, Japan).

For immunofluorescence staining, 5-μm thick OCT-embedded sections were fixed with 4% paraformaldehyde for 10 min and then blocked with 5% BSA in PBS for 30 min. Primary antibodies (1:100) were added, include Phospho-NF-κB p65 (Ser536), anti-Vimentin (sc-6260), anti-CDK9 (2316), anti-Phospho-CDK9 (Thr-186) (2549), anti-Cardiac Troponin T (cTNT, ab8295). Antibody incubations were carried out overnight at 4°C. Fluorophore-conjugated secondary antibodies were added for 1 h at room temperature for detection. Sections were counterstained with DAPI and cover-slipped. Fluorescence images were captured with an Olympus microscope. Images were quantified in a blind manner using ImageJ (version 1.38×, National Institutes of Health, Bethseda, MD, USA). For each heart sample, at least five random fields were measured.

In all immune staining experiments, the normal rabbit IgG or normal mouse IgG staining followed by secondary antibody staining was utilized to validate antibody specificity and distinguish genuine target staining from the background.

Transcriptome analyses
The transcriptome sequencing data of the heart from the patients with hypertrophic cardiomyopathy (HC, n=5) and healthy control samples (CS, n=4) were obtained from the public database (GSE89714). Raw RNA-seq data for GEO dataset GSE89714 were downloaded from the SRA database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89714) and quantified to gene-level counts using the ARCHS4 pipeline57. Gene counts were downloaded from the ARCHS4 gene expression matrix v5. Principal Component Analysis was performed using the PCA function from in the sklearn Python module. Prior to performing PCA, the raw gene counts were normalized using the logCPM method, filtered by selecting the 2500 genes with most variable expression, and finally transformed using the Z-score method. The volcano plot was performed with gene fold changes were transformed using log2 and displayed on the x axis; P-values were corrected using the Benjamini-Hochberg method, transformed using –log10, and displayed on the y axis. The Differential Gene Expression section for more information on the methods used to generate these values. The enrichment results were generated by analyzing the up-regulated gene sets using Enrichr58.

Kinase Enrichment Analysis (KEA)
Four published RNA-Seq databases from mouse models with or without pathological cardiac remodeling (GSE66630, GSE82294, GSE58455 and GSE101977) were used to analyze the up-regulated gene sets using Enrichr. The changed gene sets were further used for kinase enrichment analysis by ARCHS4_Kinases_Coexp. Significant results are determined by using a cut-off of p-value<0.01 after applying Benjamini-Hochberg correction. For the human hypertrophic heart tissue RNA-seq database (GSE89714), the enriched kinase result was also obtained by analyzing the up-regulated gene sets using Enrichr and then performing KEA_2015 analysis. Significant results are determined by using a cut-off of p-value<0.05 after applying Benjamini-Hochberg correction. The Venn diagram was drawn by using the online viewer tool: http://bioinfo.genotoul.fr/jvenn. The detail of the kinase enrichment analysis results was shown in Supplementary Data 1.

Real-time quantitative PCR
RNA was isolated from cultured cells and heart tissues using TRIZOL (Thermo Fisher, 15596026) or Total RNA Purification Kit (RN001, EZ Bioscience). PrimeScript RT reagent Kit (Takara, RR037A) was used for cDNA synthesis. Quantitative real-time PCR was performed using ABI QuantStudio6 detection system (Applied Biosystems, Thermo Fisher). Primers for genes were obtained from Thermo Fisher. Sequences are presented in Supplementary Table 2. mRNA of target genes was normalized to Actb housekeeping gene.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed using SimpleChIP enzymatic chromatin IP kit with magnetic beads (90T03, Cell Signaling Technology), with anti-NF-κB p65 (8242T), anti-RNAPII (2629), or IgG (2729). Quantitative PCR was performed with SYBR Green (QuantStudio, Thermo Fisher). Primers for Il6 promoter around the P65 binding site59 and Myc promoter around the RNAPII binding site60 were obtained from the previous reported, and are presented in Supplementary Table 2.

Co-Immunoprecipitation
For co-immunoprecipitation, proteins were extracted from animal myocardial tissue (10 mg) and cultured cells (NRVMs and 293T cells, 1 x107) samples with 0.4 mL IP lysis buffer (87787, Thermo Fisher). A portion of the lysate was retained as Input sample. Then, 200 μL protein lysate (1 mg/mL) was incubated with 20 μL protein A+G magnetic beads (HY-K0202, MCE) and the corresponding primary antibody (1 μg) or IgG on a vibrator at 4℃ overnight. Collected the beads by the magnetic separation and remove the supernatant. And then the magbeads-Ab-Ag complex was washed 5 times using 1ml PBS for each wash. Perform magnetic separation between each wash, remove supernatant and resuspend by gentle pipetting. The magnetic bead mixture was eluted with 1X loading buffer (100℃ for 10 min) for denaturing SDS-PAGE. The supernatant was used for subsequent western blot experiments.

Proximity Ligation Assay (PLA)
To assess the dynamic protein-protein interaction between CDK9 and p65, a Proximity Ligation Assay (PLA) was performed on NRVMs following angiotensin II (Ang II) stimulation for 0, 3, and 6 hours. Untreated cells (0 hour) served as the control. After stimulation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with Duolink® blocking buffer. Cells were subsequently incubated overnight at 4°C with a mixture of primary antibodies: rabbit anti-CDK9 (1:100) and mouse anti-p65 (1:100). Following washes, cells were incubated with Duolink PLA PLUS and MINUS probes for 1 hour at 37°C. Ligation and amplification steps were then carried out according to the manufacturer's instructions (Duolink® In Situ Detection Reagents). Nuclei were counterstained with DAPI. Immunofluorescence images were obtained.

Western blot analysis
Fifty micrograms of cell and tissue lysates or IP samples were separated by 10% SDS-PAGE and electro-transferred to a PVDF membranes. Membranes were blocked in Tris-buffered saline containing 0.05% Tween20 and 5% non-fat milk for 1.5 h. PVDF membranes were then incubated with specific primary antibodies. Immunoreactive bands were detected by incubating with secondary antibodies conjugated to horseradish peroxidase and enhanced chemiluminescence reagent (Bio-Rad). Densitometric quantification was performed using Image J analysis software version 1.38e. Proteins were normalized to their respective control (GAPDH for cytosolic proteins, Lamin B for nuclear fractions, and total protein for phosphorylated-form detection).

Quantitative Phosphoproteomics:
Quantitative phosphoproteomics was performed by MH BioTech Co.,Ltd (Shanghai, China). Briefly, the heart tissue samples were collected from three mice per group and pooled as one sample. The samples were preprocessed through protein extraction, protein digestion, TMT labeling, enrichment of modified peptides, fraction separation. The purified peptides were redissolved and analyzed by LC-MS/MS (Mass-Hub, Shanghai) using an Ultimate 3000 RSLCnano system (Thermo Fisher Scientific), which was online-connected to a Q Exactive HF mass spectrometer (Thermo Fisher) equipped with a Nanospray Flex ion source (Thermo Fisher). For phosphoproteomic analysis, phosphorylation of serine (S), threonine (T), and tyrosine (Y), were specified as variable modifications. A total of 1298 phosphoproteins and 2396 phosphorylated modification sites were identified. Student’s t-test was then performed to compare the two groups, using a P value of < 0.05 and a fold-change threshold of 1.2 for upregulation or 0.83 for downregulation as criteria for significance. Proteins with a FC > 1.2 were considered upregulated, while those with a FC < 0.83 were considered downregulated. KEGG and Reactome pathway enrichment analysis of NVP2-downregulated phosphoproteins according to their enrichment P-value (P < 0.05). For KEGG enrichment analysis and Reactome pathway enrichment analysis, the number of differentially expressed proteins (a) and the number of quantified proteins (b) are first calculated for each pathway. Subsequently, the total number of differentially expressed proteins (c) and the total number of quantified proteins (d) across all pathways are computed. Finally, using these four values (a, b, c, d), the Fisher's exact test P-value and enrichment fold are calculated. The enrichment fold is calculated using the formula: (a/c) / (b/d). For Protein interaction information, the PPI network was obtained from the STRING database (string-db.org). TOP 100 proteins were queried against protein interaction relationships in the STRING database to construct a network interaction diagram for these proteins. Core nodes within the protein interaction network were calculated using algorithms such as Degree, MCC, and MNC from the Cytoscope software.

Statistical analysis
All representative images were selected as the best representation of the average seen in the particular condition/treatment group. Statistical analysis was performed with GraphPad Prism 8.0 software (San Diego, CA, USA). In in vitro experiments, we assumed that the data follow a Gaussian distribution by relying on the central limit theorem. Data presented in in vitro study were representative of at least 3 independent experiments and were expressed as Mean ± SEM. The exact group size (n) for each experiment is provided and ‘n’ refers to independent biological replicates, not technical replicates. An unpaired 2-tailed Student’s t-test was applied for comparing two groups, and one-way ANOVA followed by Tukey post-hoc test was applied for comparing more than two groups. In in vivo experiments, Shapiro-Wilk normality test (P<0.05) was used to determine the adherence to a normal (Gaussian) distribution of data, except for groups with small n (n < 6). Mann-Whitney U test was used to analyze two-group comparisons and Kruskal-Wallis followed by Dunn post hoc multiple comparisons test for multi-group comparisons when the data failed normality test or small sample size (n < 6 per group). For the comparisons of two parametric data groups, an unpaired 2-tailed Student’s t-test was used. For the comparisons of more than two parametric data groups, one-way ANOVA followed by Tukey post-hoc test was used when one level is being compared, and 2-way repeated-measures ANOVA analysis with a single pooled variance and a Tukey correction for pairwise comparisons when two levels are being compared. P ˂0.05 was considered statistically significant, while P ≥ 0.05 was considered "not statistically different". Post-tests were run only if F achieved P<0.05 and there was no significant variance in homogeneity. Only within-test corrections were made in this study.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Guang Liang and colleagues described a novel role of cell-cycle dependent kinase 9 (CDK9) in cardiomyocyte remodeling and inflammation. They analyzed RNA Seq data from the existing databases and identified several kinases, including CDK9, which RNA levels increased in cardiac tissue obtained from patients with hypertrophic cardiomyopathy (HC). The authors used mouse model of HC, induced by the injection of Angiotensin II (Ang II), to show that CDK9 phosphorylation on Threonine 186 (Thr-186) was increased in HC hearts. They utilized small molecule CDK9 inhibitor, NVP-2, which selectively inhibits CDK9 at nanomolar concentrations, but also inhibits other CDKs at micromolar concentrations. Also, the author used siRNA against CDK9 and CDK9 T186 and T186E mutants, to mimic non-phosphorylated CDK9. They showed that both small molecular and siRNA inhibition of CDK9 prevents cardiomyocytes activation and enlargement by Ang II. RNA Seq analysis of cardiomyocytes treated with CDK9 inhibitors in combination with Ang II pointed to changes in NF-kB activation and specifically to Rel A/p65 NF-kB subunit and NF-kB mediated transcription changes. The authors explored the effect of CDK9 Thr-186 phosphorylation mice by delivering CDK9 shRNA by AAV9 to mice hearts, followed by the delivery of CDK9 expressing AAV9 vector. They concluded that CDK9 phosphorylation and overactivation of CDK9 lead to the heart hypertrophy. Analysis of CDK9 partners in 293T cells by proteomics analysis identified Rel-A subunit, which was shown to be phosphorylated by CDK9 in cyclin T1-dependent manner. Finally, the authors analyzed the effect of NVP-2 on mice HC induced by Ang II and found reduction of heart inflammation and remodeling. Overall, this is very packed study, with lots of data and plenty of complex in vivo experiments. However, there are many missing controls, misconnects, misinterpretations and misspells. The authors are unfamiliar with the regulation of CDK9. It is not a cell cycle regulatory kinase. CDK9 is held inactive in cytoplasm, in a non-phosphorylated form, the complex with chaperons, cdc37 and Hsp90. Its activation involves binding to cyclin T1 and transit to 7SK small RNA ribonucleotide complex (7SK snRNP). The CDK9/cyclin T1 reactivation from 7SK snRNP is likely to involve PKC. Ang II induces PKC and NFkB activation. Neither 7SK nor PKC are mentioned and explored in the current study. There is no single experiment analysis CDK9 activity which is implied many times. Some of the presented data are questionable and not convincing, i.e., transfection with siRNA or infection with shRNA followed by ectopic expression of CDK9 (see detailed comments below). The use of CDK9 inhibitor in vivo is doubtful, as CDK9 is the major cellular transcription kinase. As an example, the clinical trials with flavopiridol, which is the best known CDK9 inhibitor, are abandoned. Thus, the utility of CDK9 inhibition may be not that straight forward, especially if hear needs to be the target. Overall, all these concerns reduced the reviewer’s enthusiasm.\nMajor Critics\n1. Supplemental Fig.S1: CDK9 mRNA expression increase is minimal, less than 2-fold, in the HC hearts. Thus, this minor change, while statistically relevant, does not seem to be biologically significant and not clearly point to CDK9.\n2. Unclear how minimal changes in mRNA expression relates to the kinase activation which typically occurs and the level or phosphorylation and interaction with the regulatory or inhibitory proteins. There is no single experiment showing changes in CDK9 activity in this study.\n3. CDK9/cyclin T1 activity is regulated by its interaction with 7SK RNA and associated proteins (7SK snRNP). The authors completely overlook this important feature of CDK9. Moreover, CDK9 has nothing to do with the cell cycle, which is typically regulated by CDK4, CDK2 and CDK1.\n4. Ang II induced PKC and upregulated NF-kB which could lead to CDK9/cyclin T1 recruitment from 7SK snRNP.\n5. CDK9 Thr-186 phosphorylation is mediated by CDK7. Is there any changes in the CDK7 activity?\n6. Lane 45, “over-activated CDK9 with T186E”, you need to show that this mutation elevates CDK9 activity. Does this mutant associate with cyclin T1? In literature, any mutation of Thr-186 inhibits CDK9 activity.\n7. Lane 162, “ Thr186 phosphorylation” is required for CDK9 kinase activity, but the activity is regulated through the interaction of CDK9/ Cyclin T1 with 7SK snRNP as pointed above.\n8. Fig. 1C only shows changes in phosphorylation. You need to test CDK9 activity and prove that indeed it is elevated. This may not be the case.\n9. What is the ratio of large/ small CDK9 complex (i.e., 7SK snRNP vs low MW CDK9/cycT1) in the Ang II versus control heart-derived cardiomyocytes?\n10. Fig. 1C, phosphorylation of SRC looks even more elevated compared to CDK9 phosphorylation. The authors need to provide raw data for the quantification as CDK9 and SRC phosphorylation seem to be equally increased. Moreover, other data (i.e., Fig. 7) indicate that changes of CDK9 Thr-186 phosphorylation are minimal or absent in Ang II treated cells.\n11. It would be nice to test SRC activity, which is known to be induced by Ang II.\n12. Fig 1C and D, you need to show position of MW markers and not the theoretical MW of the detected band.\n13. Adjust all your ratios, so that you control = 1.\n14. Lane 184, “These findings indicate that CDK9 is phosphorylated by Ang II in hypertrophic heart tissues and cardiomyocytes.” – this is completely incorrect statement. Ang II is a peptide that induces SRC phosphorylation and activation of PKC. It can not phosphorylate proteins.\n15. Supplemental Fig. S4, you used 1 uM of NVP2 which would inhibit not only CDK9 (IC50 = 0.5 nm) but also CDK1/Cyc B and CDK2/Cyc A (IC50>1 uM) at this concentration. Thus, this is incorrect experiment. You need to test the effect of this NVP-2 concentration on the cell cycle, which is likely to be affected.\n16. Figs. 2A and B do not seem to align: MYH7 pattern seems to be similar in panel A, but is quite different in panel B.\n17. Fig.2E, you are missing a control with CDK9 T186E, which should show no effect of Ang II.\n18. Lane 198: “The phosphorylation sites in CDK9 contains Thr186, Ser347, Thr262, and Thr363; however, it has been reported that only T186 phosphorylation drives CDK9 activation, and phosphorylation” – this is incorrect statement. CDK9 Ser-90 and Ser-175 have been shown to regulate enzymatic activity of CDK9 and its association with cyclin T1 and CDK9/cyclin T1 activation.\n19. Lane 204: “ We next transfected the CDK9WT and CDK9T186A into the CDK9-deficient NRVMs (transfected with siCDK9) and then exposed to Ang II for 24 h.” - how do you expect to express CDK9 in the cells that are transfected with siCDK9? Without accurate controls and siRNA resistant mutants, this and following experiments are not convincing.\n20. As presented, it is unclear how the delivery of AAV9 to the heart was achieved. Was this a capsid mutant? Also, quantification of Fig. S10A is needed to prove that CDK9 expression has decreased. Again, it is unclear how you can achieve CDK9 expression in the cells expressing CDK9-targeting shRNA, as pointed above.\n21. What is the efficiency of AAV9 transduction? Clearly, it is not 100%. You need to show this, otherwise, these experiments are not convincing.\n22. Fig. 4B and Fig. S11A do not show changes in CDK9 activity as stated in the sentence on lane 257.\n23. Fig. 5C, unclear why CDK9 T186E causes the cardiomyopathy. Will CDK9 T186A prevent cardiomyopathy? Have you quantified this figure? The enzymatic activity of CDK9 T186E mutant needs to be clarified and its association with cyclin T1 needs to be shown.\n24. Fig 6, these experiment were done in 293T cells, so their relevance to cardiomyocytes is not clear. Also, unclear why only CDK9 was expressed and not CDK9 and cyclin T1. The dual expression is likely to change the outcome of this experiment. Rel A has been shown to be a substrate for CDK9. CDK9 interacts with CDC37 and Hsp 90 in the cytoplasm, where it is inactive. Thus, co-localization of CDK9 and Rel-A in the cytoplasm by itself is not proving the functional interaction.\n25. To identity CDK9 substrates in cardiomyocytes, one would do phosphoproteomic analysis in NVP-2-treated cardiomyocytes rather than IP from 293T cells that is not meant to detect the kinase substrates.\n26. The Kd for CDK9/p65 interaction is high (micromolar), so this is a weak interaction and not likely to happen physiologically.\n27. Your Fig. 7B shows that CDK9 Thr-186 phosphorylation requires cyclin T1, which is expected. Also, note that Ang II effect is minimal on CDK9 phosphorylation in your control, which contradicts your premise for this study.\n28. Fig. 7 D and E, I am not convinced that mixing siRNA and CDK9 expression vectors achieve any meaningful results.\n29. Fig. 7F, you show that Ang II induced CDK9/cyclin T1 assembly and its association with RNAPII, again as expected. And CDK9 Thr-186 phosphorylation is minimally or not affected by Ang II, contradicting your premise and conclusions.\n30. If you put CDK9, cyclin T1 and Rel-A in AlphaFold 3, will CDK9 and Rel-A still interact in the same way as in the absence of cyclin T1? If not, your model is likely to be incorrect, especially if phosphorylation does not occur .\n31. Fig. 8 shows, as expected that NVP-2 inhibits global transcription of CDK9-depdendent genes such as MYH7 and others. It also inhibits expression of p65 indicates that CDK9/cycT1 may regulate p65 expression, which is a known fact.\nMinor critics:\nLane 40, “an originally cell cycle-related kinase” – CDK9 is not involved in the regulation of cell cycle.\nLane 41, “Firstly.” Should be” “Firstly, “\nLane 98, “Despite the clinical anti-hypertrophic drugs are applied”, better “ Despite the use of anti-hypertrophic drugs”\nLane 104, “Protein tyrosine kinases …” - you talk about tyrosine kinases but focus on Ser/Thr kinase. There is a missing logic in the paragraph.\nLane 120 , “CylinT1” (twice) should be “cyclin T1”.\nLane 125, “ In addition to regulating cell cycle and proliferation” – CDK9 does not regulate the cell cycle progression.\nLane 127, “controls inflammatory gene expression in the macrophage” - if you read the paper, it says that the anti-inflammatory response is mediated by NELF and is independent from CDK9.\nLane 166, where is the basal protein level in the Fig. S1E?\nLane 254, “After construction, these mice were received…”, not clear what you meant. Is it “After injection, these mice received…”?\nLane 257, “Loss of CDK9 activity” – you never measured CDK9 activity.\nLaner 280, we do not say “mice were … killed”, should say “mice were … euthanized (or culled).”\nLane 343, “CDK9186 phosphorylation and Cyclin T1” should be “CDK9 Thr-186 phosphorylation and presence of Cyclin T1”\nLane 345, “ CDK9/cyclin T1 has been reported to bind RNAPII”, this statement and the reference 22 that you provide are both incorrect and misleading. CDK9 phosphorylates RNA PII and NELF, but its brought to the RNAPII by a coactivator such as BRD4, 7SK snRNP or Tat (in case of HIV-1).", "reviewer_2": "The manuscript by Ye et al describes how an originally described cell cycle kinase, CDK9 can promote inflammation and cardiac remodeling in Ang II-challenged cardiomyocytes in vitro and in an in vivo model of Ang II induced hypertension. Experiments showing CDK9 phosphorylation at Thr-186 in mouse hypertrophic hearts are supported by the same findings in human samples of hypertrophic cardiomyopathy, giving the study translational relevance. Through an elegant series of experiments using a mutation or overexpression of the Thr186 site that either blocks activity or over-activates activity, the authors show that the effects of CDK on cardiac remodeling are independent of its effects on cell cycle activity and that CDK9 directly binds to the p65 sub unit of NFκB which leads to its nuclear translocation and transcription of pro-hypertrophic and inflammatory genes. Furthermore, the effects depend on the presence of Cyclin T1. In general, the study is novel and the conclusions are supported by the data. There are some minor concerns that are specified below.\nI have the following critique:\n1. Do the authors have a better western blot for figure 1D. The phospho CDK 9 blot looks like a smear and the unedited version also does not show a specific band in the HC group which makes it difficult to ascertain whether there is an increase in the HC group. Additionally, the abbreviations CS and HC need to be explained earlier in panel A.\n2. Lines 184-185. The authors state that “These findings indicate that CDK9\n184 is phosphorylated by Ang II in hypertrophic heart tissues and cardiomyocytes”. The authors should consider their choice of wording. It sounds like Ang II is directly phosphorylating CDK9. Maybe consider, phosphorylated in an Ang II-dependent manner.\n3. Figure 2. The authors need to explain why they used various timepoints with Ang II treatment? The time course experiment shown in figure S2 revealed the greatest effect on CDK9 phosphorylation at 6 hrs but here the authors use 8 hrs and 24 hrs.\n4. The authors conclude from figure 3K that p65 mediates the actions of activated CDK9. However, figure 3K showed that silencing of p65 resulted in approximately a 50% down regulation of TNF, IL-1 and IL-6. Although knock down was not 100%, the authors need to be cautious in saying that it is the only mediator of CDK9 effects and perhaps speculate that other mechanisms also exist.\n5. The representative echocardiogram in figure 4C is of poor quality, especially the middle panel that does not clearly show the walls of the ventricle. The authors should consider updating this figure. In addition, do the authors have heart rate data for echo? This should be added to the table.\n6. Lines 296-298 are confusing. Please rewrite to make it clear that both cell types are used for these experiments.\n7. As it relates to figures 6D-E, Lines 304-305 say that Ang II challenge increased the CDK9-P65 interaction in a time-dependent manner. You cannot say it increased in a time dependent manner as analyses between time points was not done.\n8. The authors suggest that CDK9 directly binds to and phosphorylates p65 through an IKKβ-independent mechanism. However, data to support this is not strong. The authors should be cautious in making this statement.\n9. Human samples discrepancy – Figure 1 D shows N=3, the methods state N=2 human hearts were collected. Please clarify. Additionally, the authors do not state the sex of the patient samples.\n10. Line 285. The authors state that the “cardiomyocytes were loosely arranged”. However, figure 4D does not show that and the statement should be removed.\n11. Figure 6D shows an interaction between CDK9 and p65 even at the 0 hr time point after the addition of Ang II. Why is this? Does the interaction occur within a few minutes of the addition of Ang II or is it pre-existing? Additionally, the confocal images shown in figure 6H show a O hr time point but line 319 states that the zero time is without Ang II challenge. Please clarify.\n12. Supplement cell culture studies states that “…..plated in DMEM with 10% FBS to reduce the number of non myocytes”. I think the authors mean to say that a pre-plating step was used and non-adherent cardiomyocytes were collected afterwards. Please revise this sentence.\n13. Supplement cell apoptosis section talks about TUNEL staining but this was not shown in the manuscript. Please remove.\n14. Supplement heart tissue staining. Why was a semi quantitative method used to assess fibrosis? Also, the graphs of fibrosis just show percent so how do these measurements match?\n15. There are some previous publications that are relevant to this study that should be cited. For example, Sun et al (Front Cardiovasc Med 2022) showed that CDK9 binds and activates SGK to promote cardiac repair via the GSK/β catenin pathway while Funamoto et al (Pharmaceutics 2022) reported that pyrazole-curcumin decreased hypertrophy via disruption of the CDK9/Cyclin T1 complex.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This stutudy investigated the role of kinase activity CDK9 in cultured cardiomyocytes and murine models of Ang II-induced cardiac remodeling. The data indicated that CDK9 directly binds to NF-κB-P65, which leads to the CDK9/P65 complex nuclear translocation, P65 activation, resulting in inflammation and hypertrophic remodeling induced byAng II. All the experiments are well performed.\nMajor comments：\n1.The effects of CDK9 on cardiac hypertrophy and heart failure have been reported previously (PMID: 19757441; PMID: 15297879; PMID: 20081228; PMID: 17459355). This study lacks novelty.\n2.This study did not use cardiac-specific CDK9 knockout mice for Figure 4 and 5.\n3.The current results suggest that CDK9 induces cardiac inflammation and hypertrophic remodeling through interacting and activating P65, but it lacks the in vitro or in vivo experiments to demonstrate the effect of inhibition or knockdown of p65 on cardiac inflammation and hypertrophy in CDK9 (T186E)-infected cardiomyocytes or AAV9-CDK9 (T186E)-injected mice."}, "author_response": "We sincerely appreciate the time and effort you have dedicated to reviewing our manuscript. We have carefully addressed all of your concerns, and our point-by-point responses and corresponding revisions are detailed below. Specifically, we have made the following key clarifications and additions:\n1) Regarding CDK9 kinase activity: We wish to clarify that the term \"CDK9 activity\" used in our revised manuscript specifically refers to the phosphorylation status of CDK9 at Thr186 (p-CDK9, an indicator of its activated state). Based on our previous and newly supplemented data, Ang II robustly induces CDK9 phosphorylation at T186 site. This modification triggers its dissociation from 7sk RNA, strengthens its association with Cyclin T1, and enhances the phosphorylation of its downstream substrates, including RPB1 and P65. All these activated events are markedly suppressed upon CDK9 inhibition or knockout, confirming their CDK9 activity dependence. Furthermore, corroborative evidence from 293T cells demonstrates that the phospho-mimetic CDK9 T186E mutation specifically promotes P65 phosphorylation, directly establishing that the T186E mutation elevates CDK9 kinase activity. In summary, monitoring the phosphorylation status of direct CDK9 substrates (such as p-P65) can provide a reliable functional readout that reflects the changes in CDK9 kinase activity under various stimulatory or interventional conditions. Furthermore, we supplemented the study by examining changes in the CDK9/7SK RNA interaction under different stimulation conditions, providing additional mechanistic support for how Ang II facilitates the CDK9/P65 interaction.\n2) Regarding the NVP2 inhibitor: Second, regarding the use of CDK9 inhibitors, NVP2 is a potent and highly selective ATP-competitive inhibitor of CDK9. However, although flavopiridol mentioned by the Reviewer #1 has been reported to inhibit CDK9, it is a multi-targeted natural compound. Flavopiridol has been reported as a pan-CDK inhibitor that primarily targets CDK1/Cyc B1 (IC₅₀ = 27 nM), CDK2/Cyc E (IC₅₀ = 208 nM), CDK4/Cyc D1 (IC₅₀ = 132 nM), and CDK9/Cyc T (IC₅₀ = 11 nM) (PMID: 23908594). In addition, Mcl-1 (PMID: 20855960, PMID: 12533675) and EGFR (PMID: 12839951) have been identified as potential targets of flavopiridol, while the role of EGFR in ventricular remodeling is well established (PMID: 34270373, PMID: 32580159).. Therefore, using flavopiridol could not exclude its effects of its inhibition on other targets. To evaluate its effect on primary cardiomyocytes, we analyzed the cell cycle distribution of NRVMs by flow cytometry after 24 h of treatment with NVP2 at various concentrations. As shown in the updated Figure 6, NVP2 did not induce significant changes in the cell cycle profile of NRVMs at any of the tested doses. This is consistent with the biological characteristics of NRVMs as terminally differentiated cells, which possess very limited proliferative capacity and low basal cell cycle activity, supporting that CDK9 inhibition by NVP2 does not affect the cell cycle progression of NRVMs. Our experimental data indicate that treatment with 1 μM NVP2 effectively inhibits CDK9 phosphorylation activity, without adversely affecting NRVM morphology, viability, or cell cycle. Based on these findings, a concentration of 1 μM NVP2 was employed in all relevant cellular experiments in this study to ensure specific inhibition of CDK9 phosphorylation in cardiomyocytes.\n3) Regarding the CDK9 knockdown and overexpression experiments: Thirdly, for the contexts where we used shRNA and siRNA lines. We have provided all distinct AAV9 sequences in the supplementary materials. Our design strategy for AAV9 sequences specifically addresses the potential issue of complementation interference. To prevent the shRNA from inadvertently targeting the overexpressed transgene, we first introduced synonymous mutations into the CDK9 overexpression (OE) sequence within the region targeted by AAV9-shCDK9. Subsequently, we performed a targeted mutation at position 186. This dual-mutation approach ensures that, when both AAV9 vectors co-infect cardiomyocytes, the shRNA from AAV9-shCDK9 cannot recognize and silence the CDK9-OE transcript. Consequently, this enables the simultaneous knockdown of endogenous CDK9 and robust, specific overexpression of either CDK9 wild-type (WT), CDK9 T186A, or CDK9 T186E. As shown in Figure 4, 5, and Supplemental Fig.S10, our experimental strategy successfully enabled the knockdown of endogenous CDK9 in mouse cardiac tissue while allowing for the specific re-expression of our constructed CDK9 mutants."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a revised manuscript from Guang Liang and colleagues that identified a novel role of cell-cycle dependent kinase 9 (CDK9) in cardiomyocyte remodeling and inflammation. The authors showed that Ang II induced phosphorylation of CDK9 on Thr-186 and this led to the formation of p-CDK9/P65 complex that induced NF-κB-mediated inflammation and cardiac remodeling. The authors comprehensively responded to the critics. There are few minor remaining points that are listed below.\nMinor Critics\n1. Lane 147, “we found 46 kinases were activated in these four models. “ - in reality you observed increased levels of mRNA coding for these 46 kinases. This is not an observation of elevated kinase activities. You can probably change this as “ we found elevated mRNA expression levels for 46 kinases in these four models”.\n2. Lane 147, “Furthermore, by cross-analysis with the KEA on the RNA-Seq database of human hypertrophic heart samples, we finally identified 5 activated candidate kinase signals involved in cardiac remodeling (Figure 1A, Supplementary Figure 1A-D).” – can you explain what exactly you did? It is not clear from the Fig 1A and supplemental Fig.1.\nMisspells and minor clarifications:\nLane 43, “Loss function of CDK9” - should be “CDK9 loss of function”\nLanes 44-45, “whereas overactivated CDK9 with T186E mutation induces” - should dbe ““whereas CDK9 overactivation by T186E mutation induces’’\nLane 54, “This study provides targeting CDK9 as a potential therapeutic strategy of heart failure.” Best to rewrite, something like this “This study identifies CDK9 as a potential target in heart failure therapeutics”.\nLane 73, “si RNA” – should be “siRNA “\nLane 116, “Originally, CDK9 is an intracellular kinase “ – better without “Originally”: “CDK9 is cellular kinase”\nLane 138, “The CDK9-P65 complex represents a new mechanism” – better “ Formation of the CDK9-P65 complex represents a new mechanism”\nLane 147, you started Results with Table S2. It should be renamed to Table S1 and moved up, as your Methods follow the results.\nLane 199, “Thr186” – use “Thr-186” to be in line with the reset of the paper.\nLane 311, “Next, using Bio-Layer Interferometry (BLI) assay, a binding affinity was observed between rhCDK9 (recombinant human CDK9) and rhP65 (recombinant human P65) with a KD (the equilibrium dissociation constant) of 1.433 μmol/L (Figure 6C)” – better “Next, using Bio-Layer Interferometry (BLI) assay, a binding affinity was measured for rhCDK9 (recombinant human CDK9) and rhP65 (recombinant human P65) interaction and KD (the equilibrium dissociation constant) was determined to be 1.433 μmol/L (Figure 6C)”\nLane 318, “ As known, phospho-IKKβ (p-IKKβ) directly phosphorylates IκBα,” better “ Phosphorylated IKKβ (p-IKKβ) is known to directly phosphorylate IκBα,”\nLane 368, “For the translational perspective, we test whether” please used past tense “ For the translational perspective, we tested whether”.\nLane 469, “CKD9” change to “CDK9”.\nLane 594, Flow cytometry. Please indicate the type of instrument that you used for FACS.", "reviewer_2": "The authors have submitted a revised manuscript that has been edited to address my previous comments. Most comments have been addressed and new data has been added. I have only one remaining minor comment.\n1.In figure 6E, the authors report that the effects are still time-dependent. I disagree with this statement. The authors show that Ang II increases p65/CDK9 at 3 hrs but it appears to plateau and there is no further increase at the 6 and 9 hr time points. New statistical analyses show that the 6 hr time point is not different than the 3 hr time point and there is no difference between the 9 hr and 6 hr time points. The authors need to revise this sentence.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This study provided novel evidence that Ang II induced activation of CDK9 can directly phosphorylates P65 to drive cardiac inflammation and remodeling, and targeting CDK9 may be a potential therapeutic strategy of heart failure. All data support the conclusions. The methodology is sound. Others are OK. No further questions."}, "author_response": "Response: Thank you for your review. To identify potential kinase sets, we performed Kinase Enrichment Analysis (KEA) on mRNA-upregulated genes from the mice hypertrophic cardiomyopathy database, and found 46 kinases was activated in these four models.\nOf note, the Kinase Enrichment Analysis (KEA) is used to predict the activation status of relevant kinases associated with a set of unregulated genes. Therefore, the RNAseq-based KEA shows the activity-changed kinases, rather than the expression-changed kinases. Please further refer to the methodology and literatures on KEA.\n2. Lane 147, “Furthermore, by cross-analysis with the KEA on the RNA-Seq database of human hypertrophic heart samples, we finally identified 5 activated candidate kinase signals involved in cardiac remodeling (Figure 1A, Supplementary Figure 1A-D).” – can you explain what exactly you did? It is not clear from the Fig 1A and supplemental Fig.1.\nResponse: Thank you for your review. The specific procedures are detailed in the Methods - Kinase Enrichment Analysis (KEA) section and please see.\nMisspells and minor clarifications:\nLane 43, “Loss function of CDK9” - should be “CDK9 loss of function”\nLanes 44-45, “whereas overactivated CDK9 with T186E mutation induces” - should dbe ““whereas CDK9 overactivation by T186E mutation induces’’\nLane 54, “This study provides targeting CDK9 as a potential therapeutic strategy of heart failure.” Best to rewrite, something like this “This study identifies CDK9 as a potential target in heart failure therapeutics”.\nLane 73, “si RNA” – should be “siRNA “\nLane 116, “Originally, CDK9 is an intracellular kinase “ – better without “Originally”: “CDK9 is cellular kinase”\nLane 138, “The CDK9-P65 complex represents a new mechanism” – better “ Formation of the CDK9-P65 complex represents a new mechanism”\nLane 147, you started Results with Table S2. It should be renamed to Table S1 and moved up, as your Methods follow the results.\nLane 199, “Thr186” – use “Thr-186” to be in line with the reset of the paper.\nLane 311, “Next, using Bio-Layer Interferometry (BLI) assay, a binding affinity was observed between rhCDK9 (recombinant human CDK9) and rhP65 (recombinant human P65) with a KD (the equilibrium dissociation constant) of 1.433 μmol/L (Figure 6C)” – better “Next, using Bio-Layer Interferometry (BLI) assay, a binding affinity was measured for rhCDK9 (recombinant human CDK9) and rhP65 (recombinant human P65) interaction and KD (the equilibrium dissociation constant) was determined to be 1.433 μmol/L (Figure 6C)”\nLane 318, “ As known, phospho-IKKβ (p-IKKβ) directly phosphorylates IκBα,” better “ Phosphorylated IKKβ (p-IKKβ) is known to directly phosphorylate IκBα,”\nLane 368, “For the translational perspective, we test whether” please used past tense “ For the translational perspective, we tested whether”.\nLane 469, “CKD9” change to “CDK9”.\nLane 594, Flow cytometry. Please indicate the type of instrument that you used for FACS.\nResponse: Thank you for your review, we modified the all misspells and minor clarifications.\nReviewer #2 (Remarks to the Author):\n1.In figure 6E, the authors report that the effects are still time-dependent. I disagree with this statement. The authors show that Ang II increases p65/CDK9 at 3 hrs but it appears to plateau and there is no further increase at the 6 and 9 hr time points. New statistical analyses show that the 6 hr time point is not different than the 3 hr time point and there is no difference between the 9 hr and 6 hr time points. The authors need to revise this sentence.\nResponse: Thank you for your review. We have revised this sentence, to: “Subsequently, we examined the endogenous CDK9-P65 interaction in NRVMs by co-immunoprecipitation assay, while Ang II challenge for 3-9 hours significantly increased the CDK9-P65 interaction (Figure 6D-E).“\nReviewer #3 (Remarks to the Author):\nResponse: Thank you for your review.\nReviewer #4 (Remarks to the Author):\nResponse: Thank you for your review."}]

Decision letter