The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium requires Ca for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the LH1 complex in an engineered mutant strain. This system contained a chimeric gene cluster ( from and from ) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in ) was incorporated into the LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the LH1 complex; in contrast, α-N50 does not participate in Ca coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated LH1-RC complex and have unambiguously identified the location of Ca within this key antenna complex.

Summary Direct cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo , suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.

BACKGROUND: Because adult cardiomyocytes have little regenerative capacity, resident cardiac fibroblasts (CFs) synthesize extracellular matrix after myocardial infarction (MI) to form fibrosis, leading to cardiac dysfunction and heart failure. Therapies that can regenerate the myocardium and reverse fibrosis in chronic MI are lacking. The overexpression of cardiac transcription factors, including Mef2c/Gata4/Tbx5/Hand2 (MGTH), can directly reprogram CFs into induced cardiomyocytes (iCMs) and improve cardiac function under acute MI. However, the ability of in vivo cardiac reprogramming to repair chronic MI with established scars is undetermined. METHODS: We generated a novel Tcf21 iCre /reporter/MGTH2A transgenic mouse system in which tamoxifen treatment could induce both MGTH and reporter expression in the resident CFs for cardiac reprogramming and fibroblast lineage tracing. We first tested the efficacy of this transgenic system in vitro and in vivo for acute MI. Next, we analyzed in vivo cardiac reprogramming and fusion events under chronic MI using Tcf21 iCre /Tomato/MGTH2A and Tcf21 iCre /mTmG/MGTH2A mice, respectively. Microarray and single-cell RNA sequencing were performed to determine the mechanism of cardiac repair by in vivo reprogramming. RESULTS: We confirmed the efficacy of transgenic in vitro and in vivo cardiac reprogramming for acute MI. In chronic MI, in vivo cardiac reprogramming converted ≈2% of resident CFs into iCMs, in which a majority of iCMs were generated by means of bona fide cardiac reprogramming rather than by fusion with cardiomyocytes. Cardiac reprogramming significantly improved myocardial contraction and reduced fibrosis in chronic MI. Microarray analyses revealed that the overexpression of MGTH activated cardiac program and concomitantly suppressed fibroblast and inflammatory signatures in chronic MI. Single-cell RNA sequencing demonstrated that resident CFs consisted of 7 subclusters, in which the profibrotic CF population increased under chronic MI. Cardiac reprogramming suppressed fibroblastic gene expression in chronic MI by means of conversion of profibrotic CFs to a quiescent antifibrotic state. MGTH overexpression induced antifibrotic effects partly by suppression of Meox1, a central regulator of fibroblast activation. CONCLUSIONS: These results demonstrate that cardiac reprogramming could repair chronic MI by means of myocardial regeneration and reduction of fibrosis. These findings present opportunities for the development of new therapies for chronic MI and heart failure.

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