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This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2023A1515011278), the Guangdong Provincial Biotechnology Research Institute Self-Funded Project (GDBRI-ZL202602), and the China-Canada Academic Exchange and Cooperation on Cardiovascular Disease Models and Pathogenesis (MS202500058).

<ol>
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The authors declare that they have no competing interests.

In this study, we generated a CRISPR/Cas9-engineered Cracd-deficient mouse and set up a workflow to phenotype it after MI using conventional echocardiography, speckle&#8209;tracking strain imaging, and histology. The protocol produced knockouts with confirmed loss of CRACD protein. Cracd&#8209;deficient mice showed less ventricular dilation, better EF, and reduced fibrosis than WT controls after MI, confirming the model&#8217;s utility for studying cardiac remodeling.
The success of this model depends on several critical steps. gRNA design and in vitro validation directly affect the efficiency of zygote editing23. We use CRISPick or Benchling to select gRNAs with CRISPRater scores &#8805; 0.5 and then test cleavage efficiency with an in vitro Cas9/gRNA assay. Only gRNAs achieving &#62; 50% cleavage are microinjected. Proper LAD ligation is equally crucial: the 7&#8209;0 suture must pass 2&#8211;3 mm from the LAD origin at a depth of 0.5&#8211;1 mm to avoid puncturing the right ventricle (too deep) or failing to occlude the vessel (too shallow); immediate whitening of the anterior wall confirms occlusion. Heart rate control during echocardiography is also essential24, maintaining 450&#8211;550 bpm by adjusting isoflurane (typically 1&#8211;2%) improves reproducibility of conventional and strain measurements. Finally, for speckle&#8209;tracking analysis, visual assessment of the colour&#8209;coded mesh is essential25. The mesh must follow myocardial borders without jerking, otherwise manual refinement is needed; if quality remains poor after three attempts, the mouse is excluded.
Even when the protocol is followed meticulously, certain technical difficulties may arise. Systematic troubleshooting can resolve most of them. Low germline transmission rates (below 10%) usually point to poor donor oligonucleotide integration or weak gRNA activity26. Consequently, increasing the donor oligo to 20 ng/&#181;L, extending homology arms to 120&#8211;200 bp, and re&#8209;checking gRNA cleavage (&#62; 50%) can improve transmission. High post&#8209;MI mortality (above 30%) often comes from pneumothorax, bleeding and a large infarction27. Hence, keeping mice on continuous oxygen until they wake and using a heating pad to maintain body temperature reduces death rates noticeably. If the anterior wall does not turn pale after ligation, the LAD has probably been misidentified or the suture is too shallow. In this case, relocating the LAD (a bright red diagonal vessel) and re&#8209;passing the needle to a depth of 0.5&#8211;1 mm, sometimes with a dissecting microscope, resolve this issue. For poor speckle&#8209;tracking quality, low frame rate, fuzzy borders, or motion artifacts are the usual suspects28. Accordingly, raising the frame rate, fine&#8209;tuning gain, manually correcting the endocardial trace, and discarding cycles with arrhythmias help.
Beyond these operational considerations, the method has clear limitations. The Cracd knockout is global, so CRACD is missing in all tissues. Consequently, the observed protection cannot be attributed exclusively to cardiomyocytes, as systemic contributions may also play a role. A conditional knockout would be needed to separate cell&#8209;type&#8209;specific roles. Another limitation is the single time point at day 7; we see nothing about chronic remodeling. Therefore, later time points (4 or 8 weeks) should be added for long&#8209;term studies. Reproducibility also suffers from image quality dependence: not every ultrasound system can deliver clean endocardial borders, and operator experience matters. Thus, having a professionally trained person perform all surgeries and measurements is recommended.
In view of these limitations, it is useful to compare our approach with alternative strategies for studying gene function in post&#8209;MI remodeling. AAV&#8209;mediated gene manipulation and antisense oligonucleotides (ASOs) are two common alternatives29,30. AAV9 can deliver cardiac&#8209;specific, transient overexpression or knockdown without breeding, but its packaging limit (~4.7 kb), variable transduction efficiency, and off&#8209;target effects are drawbacks31. In contrast, our CRISPR/Cas9 knockout model offers permanent, complete loss of function, which is advantageous for long&#8209;term studies. ASOs provide reversible, dose&#8209;dependent knockdown with rapid onset, making them suitable for acute interventions; however, they need repeated dosing and lack tissue specificity32. Therefore, the constitutive knockout described here is best for initial discovery and chronic phenotyping.
Finally, the protocol can be extended well beyond CRACD. The same workflow&#8212;CRISPR/Cas9 generation of knockout mice combined with echocardiography, strain imaging, and histology&#8212;can evaluate any candidate gene in post&#8209;MI remodeling. The phenotyping framework also fits other cardiovascular disease models like transverse aortic constriction (pressure overload) or ischemia&#8209;reperfusion injury. Additionally, the tissue samples obtained from this protocol can be used for multi&#8209;omics analyses (transcriptomics, proteomics, metabolomics) to uncover molecular mechanisms. The Cracd&#8209;deficient model can serve as a tool for drug testing: compounds suspected to act through the CRACD pathway can be evaluated. Thus, this protocol provides a platform adaptable to a wide range of mechanistic and translational studies in cardiovascular research.

Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide, contributing to a significant burden on global cardiovascular health1. The long-term prognosis following MI is primarily determined by the process of left ventricular (LV) remodeling2, which involves ventricular dilation, wall thinning, and interstitial fibrosis3,4, which are key contributors to the progression to heart failure (HF). The global incidence of HF resulting from post-MI cardiac remodeling further exacerbates the burden of cardiovascular disease5. Despite these well&#8209;established clinical observations, systematic studies that use sensitive, reproducible in vivo models to assess the role of specific genes in post&#8209;MI remodeling are lacking. No protocol currently combines a CRISPR/Cas9&#8209;engineered knockout model with advanced strain imaging to evaluate a cytoskeletal regulator like CRACD. Thus, a method enabling precise genetic manipulation and high&#8209;resolution cardiac phenotyping is urgently needed.
Capping protein inhibiting regulator of actin dynamics (CRACD), also known as KIAA1211, is a regulator of actin polymerization predominantly localized in the cytosol that plays a critical role in cytoskeletal dynamics6. It positively regulates actin polymerization by binding to actin-capping proteins (CAPZA1, CAPZA2, CAPZB) and preventing them from capping to the barbed ends of actin filaments7. In epithelial tissues, CRACD plays a critical role in maintaining cell morphology and signal transduction by preserving the integrity of the actin cytoskeleton8,9. In tumor cells, its dysregulation is associated with enhanced cell metastasis and proliferation6,10. The role of CRACD in the heart has not been fully understood. Cardiac transcriptome and experimental intervention studies show that Cracd gene expression responds to pathological stress and may regulate genes related to the cytoskeletal dynamics during ventricular remodeling9,11. Furthermore, its inhibiting protein CapZ has been closely linked to cardiac pathology: the phosphorylation of CapZ regulates the growth of myofibrils during hypertrophy induced by phenylephrine12. Our early research indicates that moderate downregulation of CRACD confers protection against myocardial damage and preserves myofilament contractility following acute ischemia13. These findings raise the possibility that CRACD plays a role in maladaptive ventricular remodeling after MI. However, systematic in vivo loss&#8209;of&#8209;function studies testing whether Cracd deficiency modulates post&#8209;MI remodeling have been lacking, largely due to the absence of a validated step&#8209;by&#8209;step protocol for generating and phenotyping a Cracd&#8209;deficient mouse under ischemic stress.
Several approaches exist for generating constitutive knockout mice. Traditional homologous recombination in embryonic stem (ES) cells is precise but costly, time&#8209;consuming (12&#8211;18 months), and limited to certain mouse strains14. ENU (N&#8209;ethyl&#8209;N&#8209;nitrosourea) mutagenesis produces random point mutations requiring extensive backcrossing and sequencing15. In contrast, CRISPR/Cas9 offers distinct practical advantages for this study: (i) direct zygote injection shortens the timeline to 4&#8211;6 months; (ii) it allows deletion of a large genomic fragment (3153 bp) to ensure complete loss of function; (iii) it works directly on the C57BL/6N background without strain conversion; and (iv) it avoids a selectable marker, minimizing unintended transcriptional interference. Therefore, CRISPR/Cas9 is the most efficient choice for generating a Cracd&#8209;deficient line suitable for routine cardiovascular phenotyping. This technology has been widely used for conducting precise in vivo loss-of-function studies16,17.
Speckle&#8209;tracking strain imaging is a post&#8209;processing technique that follows the movement of natural acoustic speckles within the myocardium frame by frame, allowing the calculation of myocardial deformation parameters such as strain (fractional change in length) and strain rate (rate of deformation)18. Unlike conventional M&#8209;mode or two&#8209;dimensional echocardiography, which provides global functional indices (ejection fraction, fractional shortening, left ventricular internal diameter) and is relatively insensitive to regional wall&#8209;motion abnormalities, strain imaging can quantify segmental contractile function. This is particularly important in the early post&#8209;infarction period, where compensatory hyperkinesis of non&#8209;infarcted segments may preserve global ejection fraction despite significant regional dysfunction. Speckle&#8209;tracking offers several advantages over standard endpoints: it yields segmental strain and strain rate, displacement, and velocity, thereby detecting subtle subclinical changes19; it can identify dyssynchrony and early contractile deficits before irreversible remodeling occurs; and it directly interrogates the subendocardium, the layer most vulnerable to ischemia20. In this study, CRACD is a cytoskeletal regulator that may influence myofiber contractility at the microscopic level. Conventional cavity&#8209;based measurements could easily miss such local mechanical effects. Thus, integrating speckle&#8209;tracking analysis into this protocol enhances the ability to detect early protective effects of Cracd deficiency. Accordingly, integrating speckle&#8209;tracking into this protocol provides a sensitive and quantitative tool to evaluate post&#8209;infarction remodeling, especially during the first week after MI when early dilation and contractile dysfunction emerge21,22.
This protocol is designed for researchers investigating the functional impact of a specific gene deletion on acute (7&#8209;day) post&#8209;MI remodeling using a combination of constitutive knockout and high&#8209;resolution imaging. It is appropriate for hypothesis&#8209;generating studies where global loss of the target gene is acceptable and the main endpoint is early systolic function and structural changes. However, several limitations should be considered. First, the Cracd&#8209;deficient mouse is a global knockout; therefore, observed cardioprotective effects cannot be exclusively attributed to loss of CRACD in cardiomyocytes, as systemic contributions may also play a role. Future studies using conditional knockout strategies will be needed to achieve cardiac&#8209;specific deletion. Second, the protocol focuses on a single time point (day 7) and does not address chronic remodeling or HF progression. Longitudinal studies beyond the acute phase will be required. Third, speckle&#8209;tracking analysis demands high&#8209;quality images (clear endocardial borders) and dedicated software, which may not be available in all core facilities. Despite these limitations, the protocol provides a robust, reproducible platform for initial functional characterization of genes involved in post&#8209;MI cardiac remodeling.
This study aimed to utilize the CRISPR/Cas9 system to generate and validate a Cracd-deficient mouse model (C57BL/6N-Cracdem1 (c.538-83 to c.3352+255 del)) for investigating the impact of Cracd deficiency on post-MI cardiac remodeling and ventricular wall motion. MI was induced in both wild-type (WT, C57BL/6N) and Cracd-deficient mice, and comprehensive evaluations were performed one week after MI. Assessments included conventional echocardiographic parameters, speckle-tracking strain analysis, and histopathological evaluation. We specifically focused on the early post-MI phase (day 7) to capture initial remodeling events and to determine whether Cracd deficiency affects ventricular dilation and systolic function during the acute phase of MI. The findings are expected to provide valuable insights into the role of CRACD in modulating adverse cardiac remodeling after MI.

<table><tbody><tr><td>&lt;strong&gt;ACRISPR/Cas9&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trizma Hydrochloride Solution</td><td>Sigma-Aldrich, USA</td><td>T2663</td><td>Used for preparing DNA extraction buffer</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich, USA</td><td>539480</td><td>Used for mouse tail digestion</td></tr><tr><td>Green Taq Mix</td><td>Vazyme, China</td><td>P131</td><td>Used for PCR amplification</td></tr><tr><td>Agarose</td><td>BioFroxx, Germany</td><td>1110GR500</td><td>Used for DNA gel electrophoresis at 1&amp;ndash;2% concentration</td></tr><tr><td>DNA Marker</td><td>Thermo Scientific, USA</td><td>SM0242</td><td>Used as DNA ladder for sizing PCR products</td></tr><tr><td>TIANamp Genomic DNA Kit</td><td>Tiangen Biotech, China</td><td>DP304</td><td>Used for extracting genomic DNA from mouse tail tissue</td></tr><tr><td>MEGAshortscript&amp;trade; T7 transcription kit</td><td>Thermo Scientific, USA</td><td>AM1354</td><td>Used for in vitro transcription of gRNA</td></tr><tr><td>RNA purification kit</td><td>Zymo Research, USA</td><td>R1016</td><td>Used for purifying transcribed gRNA</td></tr><tr><td>BeyoCRISPR&amp;trade; sgRNA Screening Kit</td><td>Beyotime, China</td><td>D8412S</td><td>Used for in vitro evaluation of gRNA cleavage activity</td></tr><tr><td>RNase inhibitor</td><td>NEB, USA</td><td>M0314</td><td>Used to prevent RNA degradation during in vitro transcription</td></tr><tr><td>TaKaRa Taq</td><td>Takara Bio, Japan</td><td>R001A</td><td>Used for PCR genotyping of mouse tail DNA</td></tr><tr><td>FERTIUP&amp;reg; Mouse Sperm Preincubation Medium: PM</td><td>Cosmo Bio, Japan</td><td>KYD-002-EX</td><td>Used for sperm capacitation prior to in vitro fertilization</td></tr><tr><td>T7 ARCA mRNA Kit</td><td>NEB, USA</td><td>E2060</td><td>Used for in vitro transcription of Cas9 mRNA</td></tr><tr><td>Pregnant mare serum gonadotropin</td><td>MCE, USA</td><td>9002-70-4</td><td>Used for superovulation, administered by intraperitoneal injection</td></tr><tr><td>Human chorionic gonadotropin</td><td>ProSpec, Israel</td><td>HOR-250</td><td>Used for induction of ovulation, administered by intraperitoneal injection</td></tr><tr><td>RNase-free water</td><td>Aladdin, China</td><td>R665526</td><td>Used for RNA dissolution and PCR setup</td></tr><tr><td>Primers</td><td>Sangon Biotech, China</td><td>/</td><td>Used for genotyping</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science, China</td><td>R510-22-10</td><td>Used for induction and maintenance of anesthesia during surgery</td></tr><tr><td>Hair removal cream</td><td>Veet, France</td><td>1000207</td><td>Applied to the chest area to remove fur prior to surgery and ultrasound</td></tr><tr><td>7-0 Suture</td><td>Lingqiao, China</td><td>LQ7022380206</td><td>Used for permanent ligation of the left anterior descending (LAD) coronary artery</td></tr><tr><td>4-0 Suture</td><td>Lingqiao, China</td><td>LQ4022380412</td><td>Used for closing the skin incision after surgery</td></tr><tr><td>Anesthesia ventilator</td><td>Harvard Apparatus, USA</td><td>Tabletop</td><td>Used for delivering isoflurane and supporting respiration</td></tr><tr><td>Small animal ventilator</td><td>Taimeng, China</td><td>HX-101E</td><td>Used for providing positive pressure ventilation during open-chest surgery</td></tr><tr><td>Constant temperature heating pad</td><td>TigerGene, USA</td><td>TG-TP-GS</td><td>Used for maintaining mouse body temperature during surgery and recovery</td></tr><tr><td>Rib retractor</td><td>Shenzhen Huayon Biotech, China</td><td>LN-18-4101</td><td>Used for separating the ribs and exposing the heart for LAD ligation</td></tr><tr><td>Scissors</td><td>Shenzhen Huayon Biotech, China</td><td>18-0551</td><td>Used for making skin incisions and cutting tissue</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon, Japan</td><td>SMZ 745</td><td>Used for providing a magnified view during LAD artery ligation surgery</td></tr><tr><td>Microscopic needle holder</td><td>Shenzhen Huayon Biotech, China</td><td>18-2211</td><td>Used for manipulating the small 7-0 suture needle under the microscope</td></tr><tr><td>&lt;strong&gt;Chemicals and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ultrasonic coupling agents</td><td>Tianjin Jinya Technology Development, China</td><td>TM-100</td><td>Applied to the chest for echocardiography, ensures proper probe contact</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich, USA</td><td>V900894</td><td>Used for heart tissue fixation after harvest</td></tr><tr><td>Xylene</td><td>Macklin, China</td><td>1330-20-7</td><td>Used for paraffin removal and tissue clearing before staining</td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich, USA</td><td>H3136</td><td>Nuclear stain, used in HE staining,</td></tr><tr><td>Eosin</td><td>Sigma-Aldrich, USA</td><td>E4009</td><td>Cytoplasmic stain, used in HE staining,</td></tr><tr><td>Ponceau S</td><td>Sigma-Aldrich, USA</td><td>P3504</td><td>Used in Masson staining for cytoplasm staining</td></tr><tr><td>Phosphomolybdic acid</td><td>Sigma-Aldrich, USA</td><td>221856</td><td>Used in Masson staining as a mordant and for differentiation</td></tr><tr><td>Aniline blue solution</td><td>Sigma-Aldrich, USA</td><td>415049</td><td>Used in Masson staining for collagen fiber staining (blue)</td></tr><tr><td>CRACD antibody</td><td>Absea, China</td><td>KC-35356</td><td>Primary antibody for detecting CRACD protein by Western Blot, dilution 1:2000</td></tr><tr><td>GAPDH antibody</td><td>CST, USA</td><td>2118S</td><td>Loading control antibody for Western Blot, dilution 1:5000</td></tr><tr><td>Ethanol</td><td>Macklin, China</td><td>64-17-5</td><td>Used for dehydration and reagent preparation (70%, 80%, 95%, 100% grades)</td></tr><tr><td>&lt;strong&gt;Laboratory equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sorvall&amp;trade; Legend&amp;trade; Micro 17 Centrifuge</td><td>Thermo Scientific, USA</td><td>75002541</td><td>Used for routine sample preparation</td></tr><tr><td>PCR Thermal Cycler</td><td>Bio-Rad Laboratories, USA</td><td>1861096</td><td>Used for DNA amplification</td></tr><tr><td>Microinjection system</td><td>Eppendorf, Germany</td><td>5193000020</td><td>Used for microinjection of CRISPR/Cas9 reagents into zygotes</td></tr><tr><td>Vevo 2100 Imaging System</td><td>VisualSonics, Canada</td><td>Vevo2100</td><td>High-frequency imaging system for non-invasive assessment of cardiac function and structure post-MI</td></tr><tr><td>Light Microscope</td><td>Leica, Germany</td><td>DM2500</td><td>Used for observing HE and Masson-stained heart sections</td></tr><tr><td>&lt;strong&gt;Analysis software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health, USA</td><td>/</td><td>Used for image analysis, including area measurement, signal quantification, and processing of histological images (HE and Masson staining)</td></tr><tr><td>VevoLab 3.2.0 / VevoStrain</td><td>VisualSonics, Canada</td><td>/</td><td>VevoLab software for cardiac function analysis; VevoStrain module used for myocardial strain analysis to assess cardiac performance post-MI</td></tr><tr><td>GraphPad Prism 8</td><td>GraphPad Software, USA</td><td>/</td><td>Used for statistical analysis and graph preparation</td></tr></tbody></table>

generation and phenotypic characterization of a crispr/cas9-engineered cracd-deficient mouse model for post-myocardial infarction remodeling studies

Generation and Phenotypic Characterization of a CRISPR/Cas9-Engineered Cracd-Deficient Mouse Model for Post-Myocardial Infarction Remodeling Studies

Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide. This protocol describes a method for generating and characterizing a Cracd-deficient mouse line on the C57BL/6N background using CRISPR/Cas9 technology. Zygotes were co-injected with Cas9 mRNA, a gRNA construct, and a donor template designed to generate a 3153-bp genomic deletion. The edited allele was confirmed by PCR genotyping and Sanger sequencing. To assess the functional role of CRACD in post-MI remodeling, Cracd-deficient and wild-type (WT, C57BL/6N) mice underwent MI induced by permanent ligation of the left anterior descending coronary artery. On postoperative day 7, cardiac function and left ventricular wall motion were assessed using transthoracic echocardiography and speckle-tracking strain imaging, followed by histopathological evaluation with H&#38;E and Masson&#39;s trichrome staining. Representative results showed that Cracd-deficient mice exhibited reduced ventricular dilation and preserved systolic function compared to WT controls. This protocol provides a reliable experimental platform for mechanistic studies of CRACD in cardiac pathophysiology.

Generation of Cracd-deficient mice
A heritable mouse model carrying a targeted deficiency in the Cracd gene was generated using CRISPR/Cas9-mediated genome editing (Figure 1A, Figure 1B). To distinguish WT, heterozygous, and homozygous genotypes, a multiplex PCR was performed using three primers (F1, F2, R1) in a single reaction. Mice homozygous for the deletion (Cracd&#8209;deficient) displayed only the 421 bp band, WT mice showed only the 357 bp band (Figure 1C). Sanger sequencing of genomic DNA from Cracd-deficient mice confirmed the predicted deletion in the target gene compared to WT controls (Figure 1D). Western blot analysis further demonstrated a marked reduction in CRACD protein levels in cardiac tissues from Cracd-deficient mice compared with WT controls (Figure 1E). Together, these data confirm the successful establishment of the Cracd-deficient mouse line.
Echocardiography
Echocardiographic assessment on postoperative day 7 revealed pronounced pathological cardiac remodeling in WT mice after MI (WT-MI) compared with sham-operated controls (Figure 2A). The WT-MI group showed thinning of the interventricular septum (Figure 2B, C), significant left ventricular (LV) dilation as reflected by increased LVIDd (Figure 2D) and LVIDs (Figure 2E), and thinning of the posterior wall (Figure 2F, G). These structural alterations were associated with a pronounced decline in global systolic function, as demonstrated by significantly reduced EF (Figure 2H) and FS (Figure 2I). In contrast, Cracd-deficient mice displayed a cardioprotective phenotype following MI. LV dilation was significantly attenuated, with notably smaller LVIDd and LVIDs compared with the WT-MI group. Furthermore, these mice exhibited improved systolic thickening of the posterior wall and maintained significantly higher EF and FS values. These findings indicate that Cracd deficiency effectively attenuates post-infarction LV dilation and preserves global systolic function.
Speckle-tracking strain analysis
Left ventricular myocardial motion and deformation were further evaluated by speckle&#8209;tracking echocardiography using only parasternal long&#8209;axis views (Figure 3A). To ensure data reliability, we defined pragmatic criteria for acceptable output based on the capabilities of the VevoStrain software. Acceptable strain analysis required: (i) a clear parasternal long&#8209;axis view with endocardial borders visible throughout the cardiac cycle; (ii) a stable heart rate between 450&#8211;550 bpm during acquisition; (iii) after automatic tracking, the colour&#8209;coded mesh (activated via &#8220;toggle contour/vector/orbit line/B mode&#8221;) had to closely follow the endocardial and epicardial borders without jerking or losing contact; and (iv) strain curves had to be smooth, show consistent waveforms across segments, and lack sudden spikes or arrhythmic interruptions. Suboptimal output&#8212;most commonly erratic strain curves or inability to visualise clear motion trajectories&#8212;led to manual adjustment of endocardial or epicardial contours and re&#8209;analysis. If quality remained poor after three attempts, the mouse was excluded. Using these criteria, we obtained reliable strain data in WT&#8209;MI mice and Cracd&#8209;deficient MI mice.
Compared with the sham group, WT-MI mice exhibited significantly reduced radial velocity (Figure 3B) and longitudinal velocity (Figure 3C), as well as radial displacement (Figure 3D) and longitudinal displacement (Figure 3E), indicating impaired global myocardial motion. The absolute values of radial strain (Figure 3F), radial strain rate (Figure 3G), longitudinal strain (Figure 3H), and longitudinal strain rate (Figure 3I) were also markedly decreased, reflecting severely compromised regional myocardial deformation and function. In contrast, Cracd-deficient mice showed significant improvement in these parameters. Compared with the WT-MI group, they displayed better recovery of radial and longitudinal displacements. Furthermore, radial velocity, radial strain, and radial strain rate were maintained at higher levels, while longitudinal strain and longitudinal strain rate were significantly improved. These results suggest that Cracd deficiency not only preserves global systolic function but also enhances regional LV myocardial deformation after MI.
The speckle&#8209;tracking imaging analysis also included visualization of segmental strain distribution and cardiac motion trajectories. Parasternal long&#8209;axis B&#8209;mode images with segmented six regions (BA, MA, AA, AP, MP, BP) and color gradient mapping are displayed in Figure 4A. Cardiac motion trajectories, time&#8209;to&#8209;peak curves for radial and longitudinal strain, parametric distribution maps of strain synchrony, and three&#8209;dimensional reconstructions are presented in Figure 4B. These data further support the functional improvements observed in Cracd&#8209;deficient mice.
Histological examination
To assess the protective effect of Cracd deficiency against MI, histopathological examination was performed on heart sections from WT and Cracd-deficient mice at 7 days post-MI using H&#38;E and Masson&#39;s trichrome staining. Whole&#8209;heart longitudinal sections are shown in Figure 5A, with quantitative analysis of infarct size and collagen content presented in Figure 5B and Figure 5C, respectively. High&#8209;magnification views of the infarct zone, border zone, and remote zone are displayed in Figure 5D. In WT-MI hearts, H&#38;E staining showed pronounced left ventricular wall thinning and a well-demarcated infarct zone. Within the infarct region, extensive inflammatory cell infiltration, myocardial necrosis, and marked fibrosis with scar formation were observed. In the border zone, inflammatory infiltration, myocardial necrosis, fibrosis and cardiomyocyte hypertrophy were evident, while the remote zone displayed prominent hypertrophic changes. Corresponding Masson&#39;s trichrome staining further demonstrated extensive interstitial fibrosis within both the infarct and border zones of WT hearts following MI. In contrast, Cracd-deficient mice exhibited substantially attenuated pathological alterations, including reduced wall thinning, diminished inflammatory infiltration and myocardial necrosis, less pronounced cardiomyocyte hypertrophy, and markedly decreased interstitial fibrosis compared with WT-MI hearts. These histological findings indicate that Cracd deficiency mitigates post-infarction myocardial damage and adverse remodeling.
Successful protocol execution should yield: (i) a well&#8209;demarcated infarct zone with extensive collagen deposition in WT-MI hearts; (ii) clear cellular morphology without staining artifacts; and (iii) significantly attenuated pathology in Cracd&#8209;deficient MI hearts. Although infarct size may vary with ligation precision, the genotype&#8209;dependent difference is a reliable indicator of successful performance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/71451/71451fig1c1.jpg" /> 
Figure 1: Generation of Cracd-deficient mice. (A) Construction of Cracd-deficient mice. (B) Schematic of the Cracd gene modification strategy. (C) PCR genotyping of tail genomic DNA. WT mice show a single 357 bp band, while homozygous (Cracd&#8209;deficient) mice show a single 421 bp band. (D) Sanger sequencing of genomic DNA confirmed the predicted deletion in the Cracd locus of deficient mice compared to WT controls. (E) Western blot analysis showed significantly reduced CRACD protein levels in the hearts of Cracd-deficient mice compared to WT controls. n = 6 mice per group. Data are presented as mean &#177; SEM. Statistical significance was calculated with a two-tailed unpaired Student&#39;s t-test. <a href="https://www.jove.com/files/ftp_upload/71451/71451fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/71451/71451fig2c1.jpg" /> 
Figure 2: Echocardiographic assessment of cardiac function. (A) Representative B-mode and M-mode echocardiographic images of mouse hearts at diastole and systole. Quantitative analysis of (B) interventricular septum thickness at diastole (IVSd) and (C) systole (IVSs), (D) left ventricular internal diameter at diastole (LVIDd) and (E) systole (LVIDs), (F) left ventricular posterior wall thickness at diastole (LVPWd) and (G) systole (LVPWs), (H) ejection fraction (EF), and (I) fractional shortening (FS). n = 10 mice per group. Data are presented as mean &#177; SEM. Statistical significance was determined by one-way ANOVA followed by Tukey&#39;s multiple comparisons test. <a href="https://www.jove.com/files/ftp_upload/71451/71451fig2largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/71451/71451fig3c1.jpg" /> 
Figure 3: Speckle-tracking strain analysis. (A) Representative images of left ventricular wall motion and deformation at diastole and systole. Quantitative analyses of the following parameters: (B) radial velocity, (C) longitudinal velocity, (D) radial displacement, (E) longitudinal displacement, (F) radial strain, (G) radial strain rate, (H) longitudinal strain, and (I) longitudinal strain rate. n = 10 mice per group. Data are presented as mean &#177; SEM. Statistical significance was determined by one-way ANOVA followed by Tukey&#39;s multiple comparisons test. <a href="https://www.jove.com/files/ftp_upload/71451/71451fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/71451/71451fig4d1.jpg" /> 
Figure 4: Speckle-tracking imaging in WT-Sham, WT-MI, Cracd-deficient Sham, and Cracd-deficient MI groups at 7 days post-MI. (A) Parasternal long&#8209;axis B&#8209;mode images. Left ventricular wall was segmented into six regions: BA (basal anterior), MA (mid anterior), AA (apical anterior), AP (apical posterior), MP (mid posterior), and BP (basal posterior). Color gradient from blue to red represents increasing strain magnitude. (B) Cardiac motion trajectories; time-to-peak curves for radial and longitudinal strain values along with corresponding peak times. Parametric distribution maps of strain synchrony (blue/red indicate direction: radial &#8211; away from/toward center; longitudinal &#8211; away from/toward apex). White dots = highest velocity regions, black dots = lowest velocity. Three-dimensional reconstructions (X: time, Y: spatial position, Z: strain magnitude). <a href="https://www.jove.com/files/ftp_upload/71451/71451fig4larged1.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/71451/71451fig5c1.jpg" /> 
Figure 5: Histopathological analysis of cardiac remodeling after MI. (A) Whole-heart longitudinal sections with H&#38;E and Masson&#39;s trichrome staining. Scale bars: 2 mm. (B) Quantitative analysis of infarct size and (C) collagen content. (D) High&#8209;magnification views of infarct zone (IZ), border zone (BZ), and remote zone (RZ). H&#38;E: yellow circles = inflammatory infiltration; black arrows = myocardial necrosis; green stars = fibrosis; blue arrows = hypertrophy. Masson&#8217;s trichrome:&#160;blue indicates collagen deposition. Scale bar: 50 &#956;m. n = 5 mice per group. Data are presented as mean &#177; SEM. Statistical significance was determined by one-way ANOVA followed by Tukey&#39;s multiple comparisons test. <a href="https://www.jove.com/files/ftp_upload/71451/71451fig5largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the generation and phenotypic characterization of a C57BL/6N-Cracdem1(c.538-83 to c.3352+255 del) mouse model using CRISPR/Cas9 technology to study the role of CRACD in cardiac remodeling following myocardial infarction.

Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide. This protocol describes a method for generating and characterizing ...

We study how Cracd-deficiency influences heart remodeling and function after myocardial infarction using a Cracd-deficient mouse model. This protocol can be applied to study gene functions in post-MI cardiac remodeling by combining CRISPR knockout with vivos during imaging. To begin, use CRISPR-Cas9 gene editing technology to generate Cracd-deficient mice on a C57BL/6N background.Place the anesthetized mice in a supine position on a surgical platform and confirm deep anesthesia by the absence of the pedal withdrawal reflex. Then perform orotracheal intubation using a 20 gauge intravenous catheter sheath. Place a small tuft of cotton wool at the opening of the tube to confirm successful intubation as it moves rhythmically with the mouse's breathing.Connect the tube to a mechanical ventilator upon successful intubation to maintain ventilation and anesthesia with 2%isoflurane. Apply ophthalmic ointment to both eyes to prevent corneal drying. Then remove the hair from the anterior chest using a depilatory cream and disinfect the surgical area by applying povidone iodine and 70%alcohol multiple times.After fully exposing the skin, make a 1 to 1.5 centimeter incision in the left chest wa

generation-phenotypic-characterization-crisprcas9-engineered-cracd

Research

JoVE Journal

Medicine

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A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

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Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

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Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

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A Cryoinjury Model to Study Myocardial Infarction in the Mouse

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Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

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In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

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Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

Guangdong Provincial Biotechnology Research Institute (Guangdong Provincial Laboratory Animals Monitoring Center).  This protocol describes the generation and phenotypic characterization of a C57BL/6N-Cracdem1(c.538-83 to c.3352+255 del) mouse model using CRISPR/Cas9 technology to study the role of CRACD in cardiac remodeling following myocardial infarction.

Generation and Phenotypic Characterization of a CRISPR/Cas9-Engineered Cracd-Deficient Mouse Model for Post-Myocardial Infarction Remodeling Studies

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