Ronald J. Vagnozzi

c-kit + cells minimally contribute cardiomyocytes to the heart

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level.

Cyclophilin D controls mitochondrial pore–dependent Ca2+ exchange, metabolic flexibility, and propensity for heart failure in mice

Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif(-/-) mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif(-/-) mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif(-/-) mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif(-/-) mice was associated with an alteration in MPTP-mediated Ca(2+) efflux resulting in elevated levels of mitochondrial matrix Ca(2+) and enhanced activation of Ca(2+)-dependent dehydrogenases. Elevated matrix Ca(2+) led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca(2+) levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.

Sunitinib-induced cardiotoxicity is mediated by off-target inhibition of AMP-activated protein kinase.

Tyrosine kinase inhibitors (TKIs) are transforming the treatment of patients with malignancies. One such agent, sunitinib (Sutent, Pfizer, New York, NY, USA), has demonstrated activity against a variety of solid tumors. Sunitinib is “multitargeted,” inhibiting growth factor receptors that regulate both tumor angiogenesis and tumor cell survival. However, cardiac dysfunction has been associated with its use. Identification of the target of sunitinib‐associated cardiac dysfunction could guide future drug design to reduce toxicity while preserving anticancer activity. Herein we identify severe mitochondrial structural abnormalities in the heart of a patient with sunitinib‐induced heart failure. In cultured cardiomyocytes, sunitinib induces loss of mitochondrial membrane potential and energy rundown. Despite the latter, 5′ adenosine monophosphate‐activated protein kinase (AMPK) activity, which should be increased in the setting of energy compromise, is reduced in hearts of sunitinib‐treated mice and cardiomyocytes in culture, and this is due to direct inhibition of AMPK by sunitinib. Critically, we find that adenovirus‐mediated gene transfer of an activated mutant of AMPK reduces sunitinib‐induced cell death. Our findings suggest AMPK inhibition plays a central role in sunitinib cardiomyocyte toxicity, highlighting the potential of off‐target effects of TKIs contributing to cardiotoxicity. While multitargeting can enhance tumor cell killing, this must be balanced against the potential increased risk of cardiac dysfunction.

MICU1 motifs define mitochondrial calcium uniporter binding and activity.

Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

GSK-3α is a central regulator of age-related pathologies in mice

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Glycogen Synthase Kinase-3β Regulates Post–Myocardial Infarction Remodeling and Stress-Induced Cardiomyocyte Proliferation In Vivo

Rationale: Numerous studies have proposed that glycogen synthase kinase (GSK)-3&bgr; is a central regulator of the hypertrophic response of cardiomyocytes. However, all of this work has relied on overexpression of GSK-3&bgr;, expression of constitutively active mutants, or small molecule inhibitors with documented off-target effects. Genetic loss of function approaches have not been used in the adult mouse because germ-line deletion of GSK-3&bgr; is embryonic-lethal. Objective: This study was designed to define the role played by GSK-3&bgr; in pressure overload (PO)-induced hypertrophy and remodeling following myocardial infarction (MI). Methods and Results: We used a mouse model that allows inducible, cardiomyocyte-specific deletion of GSK-3&bgr; in the adult knockout. Surprisingly, we find that knockout mice exposed to PO induced by thoracic aortic constriction exhibit a normal hypertrophic response. Thus, in contrast to virtually all prior published studies, GSK-3&bgr; appears to play at most a minor role in the hypertrophic response to PO stress. However, GSK-3&bgr; does regulate post-MI remodeling because the GSK-3&bgr; knockouts had less left ventricular dilatation and better-preserved left ventricular function at up to 8 weeks post-MI despite demonstrating significantly more hypertrophy in the remote myocardium. Deletion of GSK-3&bgr; also led to increased cardiomyocyte proliferation following PO and MI. Conclusions: Deletion of GSK-3&bgr; protects against post-MI remodeling and promotes stress-induced cardiomyocyte proliferation in the adult heart. These studies suggest that inhibition of GSK-3&bgr; could be a strategy to both prevent remodeling and to promote cardiac regeneration in pathological states.

Cardiac Fibroblast Glycogen Synthase Kinase-3β Regulates Ventricular Remodeling and Dysfunction in Ischemic Heart

Background— Myocardial infarction–induced remodeling includes chamber dilatation, contractile dysfunction, and fibrosis. Of these, fibrosis is the least understood. After myocardial infarction, activated cardiac fibroblasts deposit extracellular matrix. Current therapies to prevent fibrosis are inadequate, and new molecular targets are needed. Methods and Results— Herein we report that glycogen synthase kinase-3&bgr; (GSK-3&bgr;) is phosphorylated (inhibited) in fibrotic tissues from ischemic human and mouse heart. Using 2 fibroblast-specific GSK-3&bgr; knockout mouse models, we show that deletion of GSK-3&bgr; in cardiac fibroblasts leads to fibrogenesis, left ventricular dysfunction, and excessive scarring in the ischemic heart. Deletion of GSK-3&bgr; induces a profibrotic myofibroblast phenotype in isolated cardiac fibroblasts, in post–myocardial infarction hearts, and in mouse embryonic fibroblasts deleted for GSK-3&bgr;. Mechanistically, GSK-3&bgr; inhibits profibrotic transforming growth factor-&bgr;1/SMAD-3 signaling via interactions with SMAD-3. Moreover, deletion of GSK-3&bgr; resulted in the significant increase of SMAD-3 transcriptional activity. This pathway is central to the pathology because a small-molecule inhibitor of SMAD-3 largely prevented fibrosis and limited left ventricular remodeling. Conclusions— These studies support targeting GSK-3&bgr; in myocardial fibrotic disorders and establish critical roles of cardiac fibroblasts in remodeling and ventricular dysfunction.

Sorafenib Cardiotoxicity Increases Mortality After Myocardial Infarction

Rationale: Sorafenib is an effective treatment for renal cell carcinoma, but recent clinical reports have documented its cardiotoxicity through an unknown mechanism. Objective: Determining the mechanism of sorafenib-mediated cardiotoxicity. Methods and Results: Mice treated with sorafenib or vehicle for 3 weeks underwent induced myocardial infarction (MI) after 1 week of treatment. Sorafenib markedly decreased 2-week survival relative to vehicle-treated controls, but echocardiography at 1 and 2 weeks post MI detected no differences in cardiac function. Sorafenib-treated hearts had significantly smaller diastolic and systolic volumes and reduced heart weights. High doses of sorafenib induced necrotic death of isolated myocytes in vitro, but lower doses did not induce myocyte death or affect inotropy. Histological analysis documented increased myocyte cross-sectional area despite smaller heart sizes after sorafenib treatment, further suggesting myocyte loss. Sorafenib caused apoptotic cell death of cardiac- and bone-derived c-kit+ stem cells in vitro and decreased the number of BrdU+ (5-bromo-2’-deoxyuridine+) myocytes detected at the infarct border zone in fixed tissues. Sorafenib had no effect on infarct size, fibrosis, or post-MI neovascularization. When sorafenib-treated animals received metoprolol treatment post MI, the sorafenib-induced increase in post-MI mortality was eliminated, cardiac function was improved, and myocyte loss was ameliorated. Conclusions: Sorafenib cardiotoxicity results from myocyte necrosis rather than from any direct effect on myocyte function. Surviving myocytes undergo pathological hypertrophy. Inhibition of c-kit+ stem cell proliferation by inducing apoptosis exacerbates damage by decreasing endogenous cardiac repair. In the setting of MI, which also causes large-scale cell loss, sorafenib cardiotoxicity dramatically increases mortality.

Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K Limits Oxidative Stress, Injury, and Adverse Remodeling in the Ischemic Heart

Blocking the activity of a cardiomyocyte-specific protein kinase with a small-molecule inhibitor reduces oxidative stress, myocyte death, and adverse remodeling in the ischemic heart. Blocking Cardiac Kinase Prevents Heart Damage Restoring blood flow after a heart attack is essential; yet, rapid reperfusion of blood can cause adverse effects on heart cells (cardiomyocytes) via oxidative damage, calcium overload, and inflammation. To limit these effects, Vagnozzi and colleagues developed an inhibitor that targets a cardiomyocyte-specific kinase called TNNI3K, which may be intimately involved in signaling events after ischemia (blockage of blood flow) and reperfusion. The authors first confirmed that TNNI3K is up-regulated in tissues from patients with heart failure who were undergoing transplant. Mice that overexpressed active TNNI3K had larger infarcts than those with an inactive form of the kinase, as well as worse ischemic injury and cardiomyocyte death. Conversely, deletion of Tnni3k reduced infarct size and prevented cardiomyocyte death in mice. From the human tissues, the kinase appeared to be limited to cardiomyocytes, which lends itself to targeted therapy. Vagnozzi et al. administered two different small-molecule inhibitors during reperfusion to mice with ischemic injury and observed a reduction in left ventricle dysfunction, progressive remodeling, and fibrosis (a hardening of the heart tissue). The authors believe that these functional benefits stem from a concomitant reduction in superoxide production, p38 activation, and infarct size. This inhibition strategy will need to be tested in a large-animal model before translation. If successful, it could find immediate application to patients with chronic ischemic cardiomyopathy, where recurrent ischemia is followed by reperfusion. Percutaneous coronary intervention is first-line therapy for acute coronary syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via reperfusion injury, a phenomenon driven in large part by oxidative stress. Therapies to limit this progression have proven elusive, with no major classes of new agents since the development of anti-platelets/anti-thrombotics. We report that cardiac troponin I–interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, promotes ischemia/reperfusion injury, oxidative stress, and myocyte death. TNNI3K-mediated injury occurs through increased mitochondrial superoxide production and impaired mitochondrial function and is largely dependent on p38 mitogen-activated protein kinase (MAPK) activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic clinical intervention. TNNI3K inhibition also preserves cardiac function and limits chronic adverse remodeling. Our findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic heart and is a tractable therapeutic target for ACS. Pharmacologic TNNI3K inhibition would be cardiac-selective, preventing potential adverse effects of systemic kinase inhibition.

Glycogen Synthase Kinase-3α Limits Ischemic Injury, Cardiac Rupture, Post–Myocardial Infarction Remodeling and Death

Background— The molecular pathways that regulate the extent of ischemic injury and post–myocardial infarction (MI) remodeling are not well understood. We recently demonstrated that glycogen synthase kinase-3&agr; (GSK-3&agr;) is critical to the hearts response to pressure overload. However, the role, if any, of GSK-3&agr; in regulating ischemic injury and its consequences is not known. Methods and Results— MI was induced in wild-type (WT) versus GSK-3&agr;(−/−) (KO) littermates by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly more left ventricular dilatation and dysfunction than WT mice. KO mice also had increased mortality during the first 10 days post-MI (43% versus 22%; P=0.04), and postmortem examination confirmed cardiac rupture as the cause of most of the deaths. In the mice that survived the first 10 days, left ventricular dilatation and dysfunction remained worse in the KO mice throughout the study (8 weeks). Hypertrophy, fibrosis, and heart failure were all increased in the KO mice. Given the early deaths due to rupture and the significant reduction in left ventricular function evident as early as 1 week post-MI, we examined infarct size following a 48-hour coronary artery ligation and found it to be increased in the KO mice. This was accompanied by increased apoptosis in the border zone of the MI. This increased susceptibility to ischemic injury–induced apoptosis was also seen in cardiomyocytes isolated from the KO mice that were exposed to hypoxia. Finally, Bax translocation to the mitochondria and cytochrome C release into the cytosol were increased in the KO mice. Conclusion— GSK-3&agr; confers resistance to ischemic injury, at least in part, via limiting apoptosis. Loss of GSK-3&agr; promotes ischemic injury, increases risk of cardiac rupture, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. These findings are in striking contrast to multiple previous reports in which deletion or inhibition of GSK-3&bgr; is protective.