Rationale: The adult cardiac extracellular matrix (ECM) is largely comprised of type I collagen. In addition to serving as the primary structural support component of the cardiac ECM, type I collagen also provides an organizational platform for other ECM proteins, matricellular proteins, and signaling components that impact cellular stress sensing in vivo. Objective: Here we investigated how the content and integrity of type I collagen affect cardiac structure function and response to injury. Methods and Results: We generated and characterized Col1a2^−/− mice using standard gene targeting. Col1a2^−/− mice were viable, although by young adulthood their hearts showed alterations in ECM mechanical properties, as well as an unanticipated activation of cardiac fibroblasts and induction of a progressive fibrotic response. This included augmented TGFβ activity, increases in fibroblast number, and progressive cardiac hypertrophy, with reduced functional performance by 9 months of age. Col1a2-loxP-targeted mice were also generated and crossed with the tamoxifen-inducible Postn-MerCreMer mice to delete the Col1a2 gene in myofibroblasts with pressure overload injury. Interestingly, while germline Col1a2^−/− mice showed gradual pathologic hypertrophy and fibrosis with aging, the acute deletion of Col1a2 from activated adult myofibroblasts showed a loss of total collagen deposition with acute cardiac injury and an acute reduction in pressure overload-induce cardiac hypertrophy. However, this reduction in hypertrophy due to myofibroblast-specific Col1a2 deletion was lost after 2 and 6 weeks of pressure overload, as fibrotic deposition accumulated. Conclusions: Defective type I collagen in the heart alters the structural integrity of the ECM and leads to cardiomyopathy in adulthood, with fibroblast expansion, activation, and alternate fibrotic ECM deposition. However, acute inhibition of type I collagen production can have an anti-fibrotic and anti-hypertrophic effect.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33^−/− mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33^f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)_A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33^f/fDBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.

BACKGROUND: TCF21 (transcription factor 21) is a bHLH (basic helix-loop-helix) protein required for the developmental specification of cardiac fibroblasts (CFs) from epicardial progenitor cells that surround the embryonic heart. In the adult heart, TCF21 is expressed in tissue-resident fibroblasts and is downregulated in response to injury or stimuli leading to myofibroblast differentiation. These findings led to the hypothesis that TCF21 regulates fibroblast differentiation in the adult mammalian heart to affect fibrosis. METHODS: Tamoxifen-inducible Cre genetic mouse models were used to permit either Tcf21 gene deletion or its enforced expression in adult CFs. Histological and echocardiographic analyses were used, as well as transcriptomic analysis to determine the consequences of TCF21 gain-of-function and loss-of-function in vivo. Genomic Tcf21 occupancy was identified by chromatin immunoprecipitation and sequencing in CFs. Myocardial infarction and AngII (angiotensin II)/phenylephrine served as models of cardiac fibrosis. RESULTS: Acute and long-term deletion of Tcf21 in CFs of the adult mouse heart does not alter fibroblast numbers, myofibroblast differentiation, or fibrosis. Fibroblast-specific Tcf21 gene–deleted mice demonstrate no significant alterations in cardiac function or scar formation in response to cardiac injury compared with control mice. In contrast, enforced expression of TCF21 in CFs inhibits myofibroblast differentiation and significantly reduces cardiac fibrosis and hypertrophy in response to 1 week of Ang II/phenylephrine infusion. Mechanistically, sustained TCF21 expression prevents the induction of genes associated with fibrosis and ECM (extracellular matrix) organization. CONCLUSIONS: TCF21 expression is not required to maintain the cell state of CFs in the adult heart. However, preventing the normal downregulation of TCF21 expression with injury reduces myofibroblast formation, cardiac fibrosis, and the acute cardiac hypertrophic response following 1 week of Ang II/phenylephrine stimulation.

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2 weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.

BACKGROUND: Calcium (Ca ²⁺ ) dysregulation is a hallmark of heart failure, impairing excitation-contraction coupling and contributing to pathological remodeling. The sarco/endoplasmic reticulum Ca ²⁺ ATPase isoform 2a (SERCA2a) mediates Ca ²⁺ reuptake into the sarcoplasmic reticulum (SR) during diastole, but its activity declines in failing hearts. DWORF (dwarf open reading frame), a newly identified cardiac microprotein, enhances SERCA2a activity and improves cardiomyocyte Ca ²⁺ cycling and contractility. SR Ca ²⁺ release also influences mitochondrial metabolism and ATP production. Here, we investigated whether DWORF overexpression improves SR Ca ²⁺ handling, augments mitochondrial Ca ²⁺ signaling, and protects against heart failure progression. METHODS: Transgenic and adeno-associated virus approaches were used to overexpress DWORF in the heart. Mice underwent transverse aortic constriction (TAC) to model pressure overload–induced heart failure. Cardiac function, mitochondrial metabolism, SR Ca ²⁺ uptake, and remodeling were assessed. RESULTS: Mitochondria from DWORF transgenic hearts displayed increased basal respiration, maximal respiration, and spare respiratory capacity, correlating with enhanced mitochondrial Ca ²⁺ uptake kinetics. Western blot analysis showed elevated levels of active PDH (pyruvate dehydrogenase) and mitochondrial Ca ²⁺ uniporter expression in DWORF transgenic hearts, supporting a role for DWORF in Ca ²⁺ -driven metabolic regulation. Similarly, MyoAAV (myo-adeno-associated virus)-mediated DWORF overexpression enhanced mitochondrial respiration and increased levels of active PDH in adult mice. Following TAC, MyoAAV-DWORF–treated mice maintained higher left ventricular function and were protected from further deterioration compared with controls. This benefit was observed when DWORF gene therapy was delivered preventively at the time of pressure overload or after heart failure was already established. DWORF gene therapy also attenuated remodeling, with lower heart weight and lung weight-to-tibia length ratios. Seahorse analysis confirmed sustained mitochondrial improvements in both treatment paradigms. CONCLUSIONS: DWORF overexpression enhances SR Ca ²⁺ dynamics, improves mitochondrial energetics, and attenuates pathological remodeling and heart failure progression in response to pressure overload. These findings support DWORF as a promising therapeutic target for heart failure.

Screening for cell surface proteins up-regulated under stress conditions may lead to the identification of new therapeutic targets. To search for genes whose expression was enhanced by treatment with oligomycin, a mitochondrial-F(0)F(1) ATP synthase inhibitor, signal sequence trapping was performed in H9C2 rat cardiac myoblasts. One of the genes identified was that for neural cell adhesion molecule (NCAM, CD56), a major regulator of development, cell survival, migration, and neurite outgrowth in the nervous system. Immunohistochemical analyses in a mouse myocardial infarction model revealed that NCAM was strongly expressed in residual cardiac myocytes in the infarcted region. Increased expression of NCAM was also found during the remodeling period in a rat model of hypertension-induced heart failure. Lentivirus-mediated knockdown of NCAM decreased the cell growth and survival following oligomycin treatment in H9C2 cells. In primary rat neonatal cardiac myocytes, NCAM was also found to be up-regulated and played a protective role following oligomycin treatment. Analyses of downstream signaling revealed that knockdown of NCAM significantly decreased the basal AKT phosphorylation level. In contrast, NCAM mimetic peptide P2d activated AKT and significantly reduced oligomycin-induced cardiomyocyte death, which was abolished by treatment with the PI3K inhibitor LY-294002 as well as overexpression of the dominant-negative AKT mutant. These findings demonstrate that NCAM is a cardioprotective factor up-regulated under metabolic stress in cardiomyocytes and augmentation of this signal improved survival.