Summary: The most common cause of cardiovascular mortality in man is the outcome from myocardial infarction. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via 1) permanent ligation of a coronary artery in the heart (myocardial infarction), 2) cryoinjury, and 3) transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).

Summary: The most common cause of cardiovascular mortality in man is the outcome from myocardial ischemic injury. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).

Summary: The most common cause of cardiovascular mortality in man is the outcome from myocardial ischemic injury. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).

Summary: Imlantable radiotelemetry devices allow for long-term continous monitoring of blood pressure and heart rate in conscious freely moving caged animals. This protocol describes the surgical implantation of a radio transmitter device in the mouse.

Summary: The most common cause of cardiovascular mortality in man is the outcome from myocardial infarction. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocoldescribes the surgical induction of myocardial injury via 1) permanent ligation of a coronary artery in the heart (myocardial infarction), 2) cryoinjury, and 3) transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).

Summary: Hypertension in man can be modeled in the mouse by introducing a constriction in the major artery (aorta) thereby obstructing outflow and increasing the afterload to the heart. This protocol describes the procedure for aortic banding aka transverse aortic constriction (TAC).

Hypertrophic cardiomyocyte growth occurs in response to various stress stimuli including biomechanical stress and neurohormonal factors. Accumulating evidence suggest that sarcomere signaling complexes play a pivotal role in the cardiomyocyte hypertrophic response by transmitting signals to the nucleus to induce gene expression. Cardiac ankyrin repeat protein (CARP, Ankrd1) is a transcriptional regulatory protein that also associates with the titin I-band spring domain, however the exact role of CARP in the heart remains to be elucidated. We report that CARP directly interacts with mitogen activated protein kinase ERK1/2 and cardiac transcription factor GATA4. Phenylephrine (PE) stimulation in cardiomyocytes induced ERK1/2 and GATA4 to transiently co-localize with sarcomeric CARP, followed by translocation of CARP and GATA4 to the nucleus. Four-and-a-half-LIM (FHL) domains proteins are part of a sarcomeric ERK2 sensory complex and knockdown of CARP by small interfering RNA (siRNA) resulted in disruption of FHL1 and FHL2. Moreover, loss of CARP attenuated PE-induced phosphorylation of ERK1/2 and GATA4, decreased GATA4 DNA binding, and prevented PE-induced cardiomyocyte growth. Mice lacking CARP have decreased FHL1 levels, and PE stimulation in wild-type mice resulted in elevated GATA4 phosphorylation and a hypertrophic response, which were completely abrogated in CARP-KO mice. We demonstrate that CARP plays an important role in PE-induced hypertrophic signaling by recruiting ERK2 and GATA4 into a titin I-band macro-molecular complex to induce GATA4 activation, followed by translocation of CARP and GATA4 to the nucleus to enhance GATA4 DNA binding and hypertrophic gene expression. Loss of CARP destabilizes FHL1 and FHL2, resulting in disruption of the PE-induced sarcomeric complex and abrogation of the cardiomyocyte hypertrophic response. These data reveal a novel role for sarcomeric titin I-band as a transcription factor activation hub that induces downstream nuclear signaling in response to agonist-induced hypertrophic stimuli.

Cardiomyocyte mechanical stretch induces hypertrophic gene expression, however, the mechanisms for this are poorly understood. Cardiac ankyrin repeat protein (CARP) is highly expressed in cardiomyocytes and interacts with the spring domain of sarcomeric titin and is also localized in the nucleus. This dual localization suggests that CARP may couple titin spring mechanics to muscle gene expression. CARP is also expressed in cardiac fibroblasts (CF) and that CARP interacts with the transcription factor GATA4. We hypothesize that CARP is a bio-mechanosensor that upon cell stretch, translocates to the nucleus and interacts with GATA4 to induce gene expression. Methods: Neonatal Rat Ventricular Myocytes (NRVMs) and CF were isolated from 1-2 day old rats and cultured on BioFlex plates. After 2 days, NRVMs were transfected with CARP siRNA (50nM) and/or a GATA4-luciferase vector. NRVMs and CF were stretched 5-10% for 60 min (S60) to 48hr at 1Hz using the Flexcell system. Cells were fixed or lysed for microscopy, western blotting, or luciferase assay. Results: Unstretched NRVMs have predominantly sarcomeric CARP immunostaining with low nuclear CARP. CARP translocates to the nucleus with S60 and remains nuclear up to 48hr stretch. ERK inhibition with U0126 prevented S-60-induced CARP nuclear translocation. NRVM S60 induced GATA4 phosphorylation and increased GATA4-luciferase expression, which are both inhibited with CARP siRNA. NRVM S60 followed by cessation of stretch for 60 min (SC60) resulted in depletion of nuclear CARP. CF show nuclear CARP, which becomes cytoplasmic with S60. CF subjected to S60 followed by SC60 showed re-localization of CARP to the nucleus. Conclusion: We conclude that CARP is involved in stretch-mediated signaling in cardiac myocytes and fibroblasts. Our data further suggest disparate mechano-sensing roles for CARP in these cells types.

Introduction: Regeneration of heart tissue post-infarction is hampered by the limited proliferation of cardiomyocytes. Due to their expandability and pluripotency, human induced pluripotent stem cells (iPSCs) are considered an ideal cell source to produce cardiomyocytes and regenerate heart tissue. However, previous clinical trials revealed the quick loss of injected cells (> 90%) after delivery in aqueous solutions (e.g. phosphate buffered saline, PBS), which significantly limited the clinical outcome. To enhance the retention of cells and promote tissue regeneration, we synthesized a temperature-responsive polymer which is water-soluble to encapsulate cardiomyocytes at room temperature, following injection into epicardium, it quickly forms a gel and holds the cells in situ at body temperature. Functional peptides can be conjugated to this material and facilitate its adhesion to cardiac tissue for optimal cell integration and cardiac regeneration. Materials and Methods: The polymers monomethoxypoly(ethylene glycol) (mPEG) and poly(ε-caprolactone) (PCL) were copolymerized to produce mPEG-PCL. The polymer was further modified with decorin-derived peptide, which can firmly bind to collagenous tissue. The solution-to-gel transition temperature (Ts) was determined by dissolving the polymer in PBS, slowly increasing the temperature from 4 to 40°C and checking the fluidity of the solution. The viability of cardiomyocytes encapsulated in polymer gel at 37°C for 2 weeks was determined by calcein AM. Peptide-modified polymer solution was injected to the epithelium of adult rat heart. Echocardiography was performed to evaluate the heart functions before and two weeks post injection. The tissue response to this material was studied by histological staining. Results and Conclusion: The material successfully underwent solution-to-gel transition at 37°C. Human iPSCs-derived cardiomyocytes encapsulated in the gel were still viable after 2 weeks. There was no heart dysfunction 2 weeks post-injection. No significant inflammatory response was induced in vivo. These demonstrate the safety and feasibility of this material to delivery cardiomyocytes to infarct heart.

Doxorubicin (adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin, however, the mechanism(s) of doxorubicin-induced CARP suppression and its specific role in cardiomyocyte biology remains to be elucidated. In this study, we report that treatment of cardiomyocytes with doxorubicin resulted in complete suppression of CARP promoter activity, decreased CARP protein levels, and marked sarcomere disarray. Transfection of CARP siRNA in cardiomyocytes resulted in a complete depletion of CARP and significant disruption of sarcomere ultrastructure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. GATA4 has previously been shown to regulate CARP, thus we examined the role of GATA4 in doxorubicin-induced CARP depletion. Cardiomyocytes treated with doxorubicin show a concomitant depletion of CARP and GATA4 protein levels. GATA4 siRNA inhibits while GATA4 overexpression enhances CARP promoter activity in cardiomyocytes. Both GATA4 and CARP siRNA significantly repressed titin and actin promoter activity. These data show that in cardiomyocytes transcription factor GATA4 is upstream of CARP and that doxorubicin induces a rapid down-regulation of GATA4 resulting in inhibition of CARP transcription. Our data further support a role for a GATA4/CARP signaling axis in sarcomere maintenance and that suppression of this pathway contributes, in part, to the overall pathophysiology of doxorubicin cardiomyopathy.

Objectives: Little is known about right ventricular (RV) remodeling in human end-stage heart failure (HF). We sought to study RV contractile protein expression in HF. Methods: Western Blot (WB) analyses were performed on protein samples prepared from the RV and left ventricle (LV) free walls of 10 explanted human hearts (5 ischemic, ISC; 5 non-ischemic, N-ISC) and two normal (NOR) donor hearts. Tissues were homogenized in lysis buffer, subjected to electrophoresis gel separation and immune-blotted with antibodies against Myosin Heavy Chain 7 (MHC), Myosin Light Chain 3 (MLC), Troponin I type 3 (TNI), Tropomyosin 1 alpha (TRP), alpha Actinin-2 (ACT) and Desmin (DSM). GAPDH served as an internal loading control. The fluorescent intensity of reactive bands was detected by Li-Cor and band densities were quantified using Image Studio software. Results: We previously identified distinct RV and LV proteomic signatures in ISC in HF and confirmed these results by WB (Figure 1). In this study focused on the RV alone, we compared contractile protein expression in NOR, ISC and N-ISC RV (Figure 2). Expression of MHC and TNI was decreased and expression of MLC, TRP and DSM increased in both ISC and N-ISC RV compared to NOR. Expression of ACT was decreased in N-ISC RV but increased in ISC RV compared to NOR. Conclusions: RV contractile proteins exhibit distinctive expression patterns in human ISC and N-ISC HF compared to normal expression patterns in controls. In HF, RV expression of MLC and TRP is significantly increased and RV expression of MHC is significantly decreased. ACT RV expression is decreased only N-ISC HF.

One of the hallmarks of aging is an increase in ventricular passive stiffness leading to diastolic heart failure, however, the molecular basis for this is unclear. The giant elastic protein titin, working as an entropic spring, is the major determinant of passive cardiomyocyte stiffness. We hypothesized that accumulation of oxidized titin in the aging heart results in increased cardiomyocyte passive stiffness and thereby contributes to diastolic dysfunction. We further hypothesized that an age-assocated disturbance in the ubiquitin/proteasome pathway plays a role in the accumulation of oxidized titin. Methods: Cardiomyocytes from adult (4 month) and aging (34 month) mouse hearts were isolated and chemically skinned and the passive force-sarcomere length relationship in single cells were obtained. Post-translational modification of titin was assessed in whole heart homogenates by determining the content of 1) titin carbonyls, a marker of irreversible protein oxidation, and 2) titin ubiquitinylation, a marker for proteasomal degradation. Results: Cardiomyocyte passive tension was significantly higher in the aging group (at SL=2.45 μm, 1008.4 ± 45.5 vs. 871.6 ± 48.3 μg; p<0.05). While the total amount of titin was unchanged between both groups, the carbonyl content of titin was increased 3-fold with aging (p<0.05). High molecular weight ubiquitin-conjugated proteins were increased with aging, which is consistent with a decrease in proteasomal activity with age. Surprisingly, titin ubiquitinylation was decreased with aging suggesting an impairment upstream of the proteasome, in titin-specific ubiquitin ligase activity. Furthermore, the muscle specific ubiquitin ligase MURF-2, which has been shown to interact with titin and play a role in muscle protein turnover, showed a significant shift from a high to low molecular weight isoform with aging. Conclusion: Our data suggests that an age-associated impairment in titin turnover contributes to accumulation of oxidatively modified titins with consequent impairment of cardiomyocyte stiffness. We speculate that the oxidation of titin will change the elastic properties of titin, directly accounting for the change in passive tension and contributing to diastolic dysfunction associated with aging.

The chemotheraputic doxorubicin has been shown to be cardiotoxic and cause myofibrillar disarray in cardiac myocytes. CARP is both a sarcomeric protein and a known transcription factor whose expression is suppressed with doxorubicin treatment. We hypothesized that disruption of CARP expression by doxorubicin is mechanistic in subsequent myofibrillar disarray.