Search

Rationale: Cardiac muscle adapts to increase workload by altering cardiomyocyte size and function resulting in cardiac hypertrophy. G protein–coupled receptor signaling is known to govern the hypertrophic response through the regulation of ion channel activity and downstream signaling in failing cardiomyocytes. Objective: Transient receptor potential canonical (TRPC) channels are G protein–coupled receptor operated channels previously implicated in cardiac hypertrophy. Our objective of this study is to better understand how TRPC channels influence cardiomyocyte calcium signaling. Methods and Results: Here, we used whole cell patch clamp of adult cardiomyocytes to show upregulation of a nonselective cation current reminiscent of TRPC channels subjected to pressure overload. This TRPC current corresponds to the increased TRPC channel expression noted in hearts of mice subjected to pressure overload. Importantly, we show that mice lacking TRPC1 channels are missing this putative TRPC current. Moreover, Trpc1 − / − mice fail to manifest evidence of maladaptive cardiac hypertrophy and maintain preserved cardiac function when subjected to hemodynamic stress and neurohormonal excess. In addition, we provide a mechanistic basis for the protection conferred to Trpc1 − / − mice as mechanosensitive signaling through calcineurin/NFAT, mTOR and Akt is altered in Trpc1 − / − mice. Conclusions: From these studies, we suggest that TRPC1 channels are critical for the adaptation to biomechanical stress and TRPC dysregulation leads to maladaptive cardiac hypertrophy and failure.

Introduction: Resident c-Kit + cardiac progenitor cells (CPCs) differentiation into cardiomyocytes in vivo is controversial. Using lineage tracing and gene ablation techniques we documented that the Wnt signaling modulator Sfrp2 substantially increased differentiation of c-Kit + CPCs into cardiomyocytes in post-MI heart and restored cardiac function. Methods: c-Kit-CreERT2/mTeG mice were used where the c-Kit promoter drives expression of a tamoxifen (TX)-inducible Cre. Upon TX treatment Cre-mediated recombination drives expression of eGFP exclusively in c-Kit cells. Mice were crossed with a Diphtheria toxin receptor (DT) strain to selectively ablate heart c-Kit cells. Mice were subjected to myocardial infarction. 2 days later, mice were injected with Sfrp2 (0.5 μg) or saline at the infarct border zone. Heart tissue was analyzed 2 months post-MI for eGFP expression and various markers by microscopy. Results: c-Kit-CreERT2/mTeG model was tested. eGFP expression had no effect on in vitro c-Kit CPC differentiation or proliferation. In TX treated mice, >90% of c-Kit+ cells were eGFP+. No eGFP+ c-Kit+ cells were seen in vehicle-only mice (N=6, p95% N=4, P=0.01). Sfrp2 induction of c-Kit differentiation to cardiomyocytes and functional improvements were completely ablated by DT. Conclusion: Using c-Kit lineage-tracing and genetic ablation we have identified that these cells are important in cardiomyocyte generation in vivo in response to Sfrp2 and improves cardiac function following MI.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a major public health problem because of its high prevalence and the current lack of effective therapies. HFpEF is thought to develop through diastolic dysfunction caused by diminished ventricular compliance, which is strongly influenced by cardiac fibrosis. Our work identifies TWIK related potassium channel-1 (TREK-1) as a novel modulator of diastolic dysfunction and cardiac fibrosis. Methods & Results: TREK-1 is expressed in mammalian cardiac tissue, although its role in cardiac function is poorly understood. To assess the role of TREK-1 in mammalian cardiac function, we interrogated cardiac function in response to pressure overload in global TREK-1 knockout (KO) mice. TREK-1 KO mice showed normal cardiac function in the basal state. However, TREK-1 KO mice developed exaggerated concentric hypertrophy in response to pressure overload. Despite developing exaggerated hypertrophy, TREK-1 KO mice maintained normal systolic and diastolic function as measured by serial echocardiography and invasive hemodynamics. Invasive hemodynamics showed preservation of ventricular compliance in pressure overloaded TREK-1 KO mice, which was associated with significantly attenuated cardiac fibrosis. The limited fibrosis in the heart was indicative of a general impairment of the fibrotic response as measured by impairments in in vivo cutaneous wound healing and in vitro fibroblast migration assays. To determine the mechanism for how TREK-1 modulates fibrosis, we investigated the activation of signaling pathways in lung fibroblasts harvested from TREK-1 KO mice. In response to the profibrotic ligands TGF beta and TNF alpha, TREK-1 KO fibroblasts showed a significantly attenuated activation of the stress-activated kinases p38 alpha and c-Jun N-terminal kinase (JNK), known mediators of myofibroblast activation. These data suggest a critical role for TREK-1 in fibroblast function via the activation of stress-activated kinases. Conclusions: We identified TREK-1 as being critical to the development of diastolic dysfunction and chamber compliance by modulating the signaling pathways involved in the tissue fibrotic response.

The DNA-binding transcription factor Nuclear Respiratory Factor 1 (NRF1) regulates mitochondrial homeostasis. Its constitutive ablation in the mouse is embryonically lethal (~E3.5). This has limited our understanding of NRF1 functionality in the heart, where mitochondrial dysfunction is often a major pathogenic factor. Therefore, we generated conditional cardiomyocyte-specific NRF1 knockout mice (MYH6-mer-Cre-mer-NRF1fl/fl or NRFfl/fl) to elucidate the role of cardiac NRF1. Two weeks after NRF1 silencing, echocardiography of NRF1fl/fl hearts revealed significant reductions in left ventricular fractional shortening (Figure A). Histology demonstrated degradation of cellular structural integrity and nuclear condensation (Figure B), with a high number of TUNEL positive nuclei compared to littermate controls (MYH6-mer-Cre-mer-NRF-1wt), indicative of apoptosis (37.8% vs. 1.1%, p < 0.001). The mRNA and protein levels of key mediators of mitochondrial biogenesis were evaluated by real-time RT-PCR and immunoblotting (Figure C & D). Compared to littermate controls, there was down-regulation of the mitochondrial encoded NADH dehydrogenase 1, implying a reduction of functional mitochondrial mass. Key biogenesis regulators PGC1-α (protein only), Nfe2l2, and NRF2 were also reduced. In total, these data support that dysregulation of mitochondrial biogenesis after loss of NRF1 results in cardiomyocyte apoptosis and reduced left ventricular function. These findings and further delineation of the mechanisms involved should lay the foundation for the exploitation of NRF1 as a therapeutic target in heart failure.

Background —Transgenic mice with constitutive myocardium-targeted expression of a peptide inhibitor of the β-adrenergic receptor kinase (βARKct) have increased in vivo cardiac function and enhanced β-adrenergic receptor (βAR) responsiveness. Methods and Results —In the present study, we created transgenic mice with myocardium-targeted βARKct transgene expression under control of the CARP (cardiac ankyrin repeat protein) promoter, which is active during cardiac development and inactive in the normal adult mouse heart. Consistent with this, adult CARP-βARKct transgenic mice have normal in vivo cardiac contractility and βAR responsiveness indistinguishable from their nontransgenic littermates (NLCs). However, because CARP is in a group of fetal genes activated in the adult ventricle during hypertrophy, we subjected animals to transverse aortic constriction (TAC) to induce pressure overload. Seven days after TAC, CARP-βARKct hearts had elevations in left ventricular mass similar to those in NLCs; however, TAC did induce demonstrable βARKct expression in the transgenic hearts. TAC in NLC mice resulted in an upregulation of myocardial βARK1 and a loss of βAR-mediated inotropic reserve. Importantly, although βARK1 was increased in the hypertrophic CARP-βARKct mice, the in vivo loss of βAR responsiveness was not seen after induced βARKct expression. Conclusions —These results demonstrate that acute βARK1 inhibition can restore lost myocardial βAR responsiveness and inotropic reserve in vivo. Furthermore, these mice demonstrate the novel utility of the CARP promoter as an inducible element responsive to pathophysiological conditions in the adult heart.

Background —Growth hormone (GH) improves cardiac function in the rat with myocardial infarction, but its effects in a model of primary dilated cardiomyopathy have not been reported. GH effects were examined at early (4 months) and late (10 months) phases of disease in the cardiomyopathic (CM) hamster, and the combination of GH with chronic ACE inhibition was assessed in late-phase heart failure. Methods and Results —CM hamsters (CHF 147 line) at 4 months showed severe systolic left ventricular (LV) dysfunction with normal LV filling pressure, and at 10 months there was more severe systolic as well as diastolic dysfunction with increasing myocardial fibrosis. Recombinant human GH alone for 3 weeks at age 4 months increased LV wall thickness and reduced systolic wall stress without altering diastolic wall stress, whereas at 10 months, wall stress and fractional shortening did not improve. The LV dP/dt max was enhanced at both ages by GH, which at 4 months reflected increased contractility, but at 10 months was most likely caused by elevation of the LV filling pressure. The increasing degree of fibrosis correlated inversely with LV function but was unaffected by GH. In other CM hamsters, high-dose ACE inhibition alone (quinapril), started at 8 months and continued for 11 weeks, improved LV function and inhibited unfavorable remodeling, but the addition of GH for 3 weeks at age 10 months produced increased wall thickness with little additional functional benefit and increased the LV filling pressure and diastolic wall stress. Conclusions —GH treatment alone improved LV dysfunction at 4 months of age in CM hamsters by increasing contractility and reducing wall stress but had few beneficial effects at 10 months in severe LV failure. After chronic ACE inhibition, addition of GH at 10 months had no additional beneficial effects and further increased LV diastolic pressure. These differing effects of GH may relate to the progressive increase of LV fibrosis in the CM hamster.

A total of 847 inbred Lewis rats of mixed sex were used in this pancreaticoduodenal (Pd) donor aging study. Pd grafts were taken from 9- to 12-month-old donors and transplanted into 3-month-old recipients (thus, the first generation Pd graft, or 1 Pd). After 9 to 12 months, the same Pd grafts were again harvested and transplanted into 3-month-old rats (thus the 2 Pd generation). This cycle was repeated to obtain the 3, 4, and 5 Pd series. Sequential transplantation was able to extend the Pd grafts' mean survival time to 32 months for fourteen 4 Pd grafts, and to 39.2 months for four 5 Pd grafts (the longest lived graft survived for 42 months). The pancreas and duodenal sections of the grafts remained normal throughout the entire study. However, the aortic sections of the grafts (which were harvested to include the superior mesenteric and celiac arteries) all exhibited moderate to massive atherosclerotic changes by the 5 Pd mean survival age of 39.2 months. Such histological changes commenced even before 21 months of Pd graft age in some animals, gradually progressing to dilation of the aorta (and subsequent narrowing of aortic tributaries), as well as formation of an eggshell-like inner membrane shielding the aortic intima, by 42 months. Such atherosclerotic changes precluded transplantations beyond the 5 Pd series.

MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary transcripts (pri-miRs), they are processed in the nucleus by Drosha into hairpin intermediates (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes following activation by a variety of signals such as those stimulated by β-adrenergic receptors (βARs). Initially discovered to desensitize βAR signaling, β-arrestins are now appreciated to transduce multiple effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the β-arrestin-biased βAR agonist carvedilol activates cellular pathways in the heart. Here, we tested the hypothesis that carvedilol could activate β-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. In human cells and mouse hearts, carvedilol (Carv) upregulates a subset of mature and pre-miRs but not their pri-miRs in a β1AR-, G protein-coupled receptor kinase (GRK) 5/6- and β-arrestin1-dependent manner (see figure). Mechanistically, β-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1, a component of the Drosha microprocessor complex (see figure). In conclusion, our findings indicate a novel function for β1AR-mediated β-arrestin1 signaling in miR biogenesis, which may be linked to its cell survival mechanism and beneficial adaptive remodeling in the failing heart.

Cardiac Troponin I-interacting kinase (TNNI3K) is a cardiac specific kinase whose biological function remains largely unknown. We have recently shown that TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of cardiomyopathy, indicating an important role in modulating disease progression. To further investigate TNNI3K kinase activity in vivo, we have generated transgenic mice expressing both wild-type and kinase-dead versions of the human TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity induces mouse cardiac hypertrophy, and the kinase activity is required to accelerate disease progression in a left-ventricular pressure overload model of mouse cardiomyopathy. We demonstrate the clinical relevance of these observations by identifying two potential missense mutations near the kinase activation loop of TNNI3K in idiopathic dilated cardiomyopathy (DCM) human patients. Using an in vitro kinase assay and proteomics analysis, we show that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity. Using antisera to TNNI3K, we show that TNNI3K protein is located at the sarcomere Z disc. These combined data suggest that TNNI3K mediates cell signaling to modulate cardiac response to stress. The essential role of the kinase activity makes TNNI3K a strong potential pharmaceutical target of kinase inhibitors for heart disease.

Transient receptor potential canonical (TRPC) channels are non-selective cation channels that are activated in response to G-protein coupled receptor activation, depletion of internal stores and mechanical stretch. Recent reports suggest that cardiac TRPC channels mediate calcineurin dependent cardiac hypertrophy, yet few details exist as to the mechanism for activation of these channels. Here, we provide evidence that TRPC1 channels are the dominant TRPC channel in mouse cardiomyocytes and cardiac TRPC1 protein expression is augmented by seven fold following thoracic aortic banding (TAC). In addition, we provide the first loss of function studies to show that mice lacking TRPC1 channels developed significantly less cardiac hypertrophy following pressure overload induced by thoracic aortic banding suggesting that TRPC1 may confer deleterious calcium entry. Whole cell voltage clamp studies of isolated adult cardiomyocytes reveal a non-selective cation current that is induced by pressure overload that is absent in TRPC1−/− cardiomyocytes and in which TRP blockers such as gadolinium, 2-amino biphenyl boric acid and SKF96365 inhibit the TAC induced current. Finally, neonatal cardiomyocytes lacking functional TRPC1 display reduced TRPC current in response to cell stretch or angiotensin-II; the functional consequence of which includes reduced calcium oscillation frequency and reduced BNP expression. These results provide the first loss of function evidence for TRPC1 channels in cardiac hypertrophy and implicate TRPC1 as a stretch activated channel.